Non-antiviral therapeutic options are required for the treatment of hospitalised patients with COVID-19. CD24Fc is an immunomodulator with potential to reduce the exaggerated inflammatory response to ...tissue injuries. We aimed to evaluate the safety and efficacy of CD24Fc in hospitalised adults with COVID-19 receiving oxygen support.
We conducted a randomised, double-blind, placebo-controlled, phase 3 study at nine medical centres in the USA. Hospitalised patients (age ≥18 years) with confirmed SARS-CoV-2 infection who were receiving oxygen support and standard of care were randomly assigned (1:1) by site-stratified block randomisation to receive a single intravenous infusion of CD24Fc 480 mg or placebo. The study funder, investigators, and patients were masked to treatment group assignment. The primary endpoint was time to clinical improvement over 28 days, defined as time that elapsed between a baseline National Institute of Allergy and Infectious Diseases ordinal scale score of 2–4 and reaching a score of 5 or higher or hospital discharge. The prespecified primary interim analysis was done when 146 participants reached the time to clinical improvement endpoint. Efficacy was assessed in the intention-to-treat population. Safety was assessed in the as-treated population. This study is registered with ClinicalTrials.gov, NCT04317040.
Between April 24 and Sept 22, 2020, 243 hospitalised patients were assessed for eligibility and 234 were enrolled and randomly assigned to receive CD24Fc (n=116) or placebo (n=118). The prespecified interim analysis was done when 146 participants reached the time to clinical improvement endpoint among 197 randomised participants. In the interim analysis, the 28-day clinical improvement rate was 82% (81 of 99) for CD24Fc versus 66% (65 of 98) for placebo; median time to clinical improvement was 6·0 days (95% CI 5·0–8·0) in the CD24Fc group versus 10·0 days (7·0–15·0) in the placebo group (hazard ratio HR 1·61, 95% CI 1·16–2·23; log-rank p=0·0028, which crossed the prespecified efficacy boundary α=0·0147). 37 participants were randomly assigned after the interim analysis data cutoff date; among the 234 randomised participants, median time to clinical improvement was 6·0 days (95% CI 5·0–9·0) in the CD24Fc group versus 10·5 days (7·0–15·0) in the placebo group (HR 1·40, 95% CI 1·02–1·92; log-rank p=0·037). The proportion of participants with disease progression within 28 days was 19% (22 of 116) in the CD24Fc group versus 31% (36 of 118) in the placebo group (HR 0·56, 95% CI 0·33–0·95; unadjusted p=0·031). The incidences of adverse events and serious adverse events were similar in both groups. No treatment-related adverse events were observed.
CD24Fc is generally well tolerated and accelerates clinical improvement of hospitalised patients with COVID-19 who are receiving oxygen support. These data suggest that targeting inflammation in response to tissue injuries might provide a therapeutic option for patients hospitalised with COVID-19.
Merck & Co, National Cancer Institute, OncoImmune.
Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense, and worsens airway inflammation. Inhaled Optate is designed to safely raise airway surface pH and is ...well tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms.
We grew primary NHAE cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether Optate would prevent viral infection and replication. Cells were pretreated with Optate or placebo prior to infection (multiplicity of infection = 1), and viral replication was determined with plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial.
Optate almost completely prevented viral replication at each time point between 24 h and 120 h, relative to placebo, on both plaque assay and N protein expression (
< .001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs, or oxygenation.
Inhaled Optate may be well suited for a clinical trial in patients with pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regard to pH, isotonicity, and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.
Trypanosoma cruzi is an intracellular parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic T. cruzi protection. We ...evaluated the importance of CCR5 and CCL5 for mucosal protection against natural oral and conjunctival T. cruzi challenges. T. cruzi-immune CCR5(-/-) and wild-type C57BL/6 mice were generated by repeated infectious challenges with T. cruzi. CCR5(-/-) and wild-type mice developed equivalent levels of cellular, humoral, and protective mucosal responses. However, CCR5(-/-)-immune mice produced increased levels of CCL5 in protected gastric tissues, suggesting compensatory signaling through additional receptors. Neutralization of CCL5 in CCR5(-/-)-immune mice resulted in decreased mucosal inflammatory responses, reduced T. cruzi-specific Ab-secreting cells, and significantly less mucosal T. cruzi protection, confirming an important role for CCL5 in optimal immune control of T. cruzi replication at the point of initial mucosal invasion. To investigate further the mechanism responsible for mucosal protection mediated by CCL5-CCR5 signaling, we evaluated the effects of CCL5 on B cells. CCL5 enhanced proliferation and IgM secretion in highly purified B cells triggered by suboptimal doses of LPS. In addition, neutralization of endogenous CCL5 inhibited B cell proliferation and IgM secretion during stimulation of highly purified B cells, indicating that B cell production of CCL5 has important autocrine effects. These findings demonstrate direct effects of CCL5 on B cells, with significant implications for the development of mucosal adjuvants, and further suggest that CCL5 may be important as a general B cell coactivator.
The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of ...TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.
The potential use of the Trypanosoma cruzi metacyclic trypomastigote (MT) stage-specific molecule glycoprotein-82 (gp82) as a vaccine target has not been fully explored. We show that the opsonization ...of T. cruzi MT with gp82-specific antibody prior to mucosal challenge significantly reduces parasite infectivity. In addition, we investigated the immune responses as well as the systemic and mucosal protective immunity induced by intranasal CpG-adjuvanted gp82 vaccination. Spleen cells from mice immunized with CpG-gp82 proliferated and secreted IFN-γ in a dose-dependent manner in response to in vitro stimulation with gp82 and parasite lysate. More importantly, these CpG-gp82-immunized mice were significantly protected from a biologically relevant oral parasite challenge.
The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously ...described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.
Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, ...we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine‐sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full‐length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.
Trypanosoma cruzi
is an intracellular parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic
T. cruzi
protection. We ...evaluated the importance of CCR5 and CCL5 for mucosal protection against natural oral and conjunctival
T. cruzi
challenges.
T. cruzi
-immune CCR5
−/−
and wild-type C57BL/6 mice were generated by repeated infectious challenges with
T. cruzi
. CCR5
−/−
and WT mice developed equivalent levels of cellular, humoral and protective mucosal responses. However, CCR5
−/−
-immune mice produced increased levels of CCL5 in protected gastric tissues, suggesting compensatory signaling through additional receptors. Neutralization of CCL5 in CCR5
−/−
-immune mice resulted in decreased mucosal inflammatory responses, reduced
T. cruzi
-specific antibody secreting cells, and significantly less mucosal
T. cruzi
protection, confirming an important role for CCL5 in optimal immune control of
T. cruzi
replication at the point of initial mucosal invasion. To further investigate the mechanism responsible for mucosal protection mediated by CCL5-CCR5 signaling, we evaluated the effects of CCL5 on B cells. CCL5 enhanced proliferation and IgM secretion in highly purified B cells triggered by suboptimal doses of LPS. In addition, neutralization of endogenous CCL5 inhibited B cell proliferation and IgM secretion during stimulation of highly purified B cells, indicating that B cell production of CCL5 has important autocrine effects. These findings demonstrate direct effects of CCL5 on B cells, with significant implications for the development of mucosal adjuvants, and further suggest that CCL5 may be important as a general B cell co-activator.
Abstract
Trypanosoma cruzi is an intracellular obligate parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic responses ...against T. cruzi. We evaluated whether CCR5 plays a critical role in mucosal protection against natural oral and conjunctival challenges. Memory-immune CCR5-/- and wild-type C57BL/6 mice were generated by repeated infectious challenges with sublethal doses of insect-derived metacyclic trypomastigotes. Similar IgA, IgG and IFN-γ responses were detected in the spleen in both memory-immune and primary infected wild-type and CCR5-/- mice. More importantly, protection was evaluated in memory-immune mice via real-time PCR and limiting dilution assay for parasite outgrowth. CCR5-/- memory-immune mice developed equivalent levels of protection against both oral and conjunctival mucosal challenges. Preliminary studies from the stomachs of CCR5-/- memory-immune mice showed an increase in the transcript levels of CCL5 as compared to memory-immune wild-type mice. Overall, it appears that CCR5 does not play a critical role in the control of parasite replication and immune responses at sites of initial mucosal infection with T. cruzi. However, CCL5 signaling through CCR1 and/or CCR3 may compensate for the absence of CCR5.
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