Case series: Maraviroc and pravastatin as a therapeutic option to treat long COVID/Post-acute sequelae of COVID (PASC).

Post-acute sequelae of COVID (PASC), or long COVID, is a multisystem complication of SARS-CoV-2 infection that continues to debilitate millions worldwide thus highlighting the public health importance of identifying effective therapeutics to alleviate this illness. One explanation behind PASC may be attributed to the recent discovery of persistent S1 protein subunit of SARS-CoV-2 in CD16+ monocytes up to 15 months after infection. CD16+ monocytes, which express both CCR5 and fractalkine receptors (CX3CR1), play a role in vascular homeostasis and endothelial immune surveillance. We propose targeting these receptors using the CCR5 antagonist, maraviroc, along with pravastatin, a fractalkine inhibitor, could disrupt the monocytic-endothelial-platelet axis that may be central to the etiology of PASC. Using five validated clinical scales (NYHA, MRC Dyspnea, COMPASS-31, modified Rankin, and Fatigue Severity Score) to measure 18 participants' response to treatment, we observed significant clinical improvement in 6 to 12 weeks on a combination of maraviroc 300 mg per oral twice a day and pravastatin 10 mg per oral daily. Subjective neurological, autonomic, respiratory, cardiac and fatigue symptoms scores all decreased which correlated with statistically significant decreases in vascular markers sCD40L and VEGF. These findings suggest that by interrupting the monocytic-endothelial-platelet axis, maraviroc and pravastatin may restore the immune dysregulation observed in PASC and could be potential therapeutic options. This sets the framework for a future double-blinded, placebo-controlled randomized trial to further investigate the drug efficacy of maraviroc and pravastatin in treating PASC.

Profiling viral receptors in nasal and amniotic samples at birth

Introduction/Aim: Children with wheeze and asthma present with airway epithelial vulnerabilities, such as impaired responses to viral infection. It is postulated that the in utero environment may contribute to the development of such airway epithelial vulnerabilities, that may predispose children to wheeze and asthma outcomes. To explore developmental mechanisms, further research is required using epithelial samples at birth. Our study asked whether amniotic epithelial samples from placentas show similar viral receptor expression to nasal epithelial cells at birth. We aimed to investigate expression of respiratory viral receptors for human rhinovirus (HRV), respiratory syncytial virus (RSV) and COVID-19-causing coronavirus (SARS-CoV-2) in nasal and amniotic epithelial samples. Methods: Unmatched nasal (n = 20 births) and amniotic (n = 33 newborns) epithelial samples were collected from ORIGINS cohort participants recruited into the AERIAL study. Using purified RNA, receptor expression for HRV (ICAM-1, LDLR, CDHR3), RSV (NCL, TLR4) and SARSCoV- 2 (ACE2, TMPRSS2) was assessed by qPCR. In addition, receptor protein expression was quantified through western blot and localized using immunohistochemistry in amniotic samples only. Results: Nasal epithelial and amniotic samples expressed various receptors for HRV, RSV and SARS-CoV-2 at the gene level in nasal (median(IQR) arbitrary units (AU);ICAM-1: 11.44(63.18);LDLR: 4.00(7.32);CDHR3: 0.40 (1.14);NCL: 2.32(2.18);CX3CR1: 2.17(2.33);TLR4: 2.20 (6.20);TMPRSS2: 1.99(4.85);ACE2: 0.36(0.52) AU) and amnion (ICAM-1: 0.69(2.21);LDLR: 0.39(1.38);CDHR3: 1.0 x 10-4(3.0x10-4);NCL: 1.03(0.55);CX3CR1: 0.12(0.24);TLR4: 0.10(0.13);TMPRSS2: 3.0 x 10-4 (16.0x10-4);ACE2: 0.01(0.02) AU). Amniotic samples also expressed these receptors at the protein level (ICAM-1: 0.03(0.05);LDLR: 0.06(0.03);CDHR3: 0.28(0.15);NCL: 0.96(1.19);CX3CR1: 0.08(0.08);TMPRSS2: 0.09(0.06);ACE2: 0.34(0.92) AU) and expression within the amniotic epithelium was confirmed by immunohistochemistry. Conclusion: Newborn nasal and amniotic epithelial samples expressed receptors for respiratory viruses, HRV, RSV, SARS-CoV-2. These findings warrant further investigation of the clinical significance of receptor expression in relation to prenatal and postnatal exposures, as well as childhood asthma development.

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity.

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5'-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5'-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.

Polyclonal expansion of TCR VBETA 21.3+ CD4+ and CD8+ T CELLS is a hallmark of multisystem inflammatory syndrome in children

Introduction: At the end of April 2020, European clinicians warned the Public Health Agencies about an abnormal increase of Kawasakilike diseases and myocarditis requiring critical care support in the context of the ongoing COVID-19 epidemic in children. American clinicians also reported a large outbreak of severe inflammation in children following COVID-19 infection, a condition that is now named Pediatric Inflammatory Multisystemic Syndrome (PIMS) or Multisystem Inflammatory Syndrome in children (MIS-C). Objectives: As MIS-C combines clinical features of Kawasaki disease (KD) and Toxic Shock Syndrome (TSS), we aimed to compare the immunological profile of pediatric patients with these different conditions. Methods: We analysed blood cytokine expression, and the T cell repertoire and phenotype in 36 MIS-C cases, which were compared to 16 KD, 58 TSS, and 42 COVID-19 cases. Results: We observed an increase of serum inflammatory cytokines (IL-6, IL-10, IL-18, TNF-a, IFNg, CD25s, MCP1, IL-1RA) in MIS-C, TSS and KD, contrasting with low expression of HLA-DR in monocytes. We detected a specific expansion of activated T cells expressing the Vβ21.3 T cell receptor β chain variable region in both CD4 and CD8 subsets in 75% of MIS-C patients and not in any patient with TSS, KD, or acute COVID-19;this correlated with the cytokine storm detected. The T cell repertoire returned to baseline within weeks after MIS-C resolution. Vβ21.3+ T cells from MIS-C patients expressed high levels of HLA-DR, CD38 and CX3CR1 but had weak responses to SARS-CoV- 2 peptides in vitro. Consistently, the T cell expansion was not associated with specific classical HLA alleles. Conclusion: Thus, our data suggested that MIS-C is characterized by a polyclonal Vβ21.3 T cell expansion not directed against SARS-CoV-2 antigenic peptides, which is not seen in KD, TSS and acute COVID-19.

Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.

The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

Acquired decrease of the C3b/C4b receptor (CR1, CD35) and increased C4d deposits on erythrocytes from ICU COVID-19 patients.

In order to study the mechanisms of COVID-19 damage following the complement activation phase occurring during the innate immune response to SARS-CoV-2, CR1 (the regulating complement activation factor, CD35, the C3b/C4b receptor), C4d deposits on Erythrocytes (E), and the products of complement activation C3b/C3bi, were assessed in 52 COVID-19 patients undergoing O2 therapy or assisted ventilation in ICU units in Rheims France. An acquired decrease of CR1 density on E from COVID-19 patients was observed (Mean = 418, SD = 162, N = 52) versus healthy individuals (Mean = 592, SD = 287, N = 400), Student's t-test p < 10-6, particularly among fatal cases, and in parallel with several parameters of clinical severity. Large deposits of C4d on E in patients were well above values observed in normal individuals, mostly without concomitant C3 deposits, in more than 80% of the patients. This finding is reminiscent of the increased C4d deposits on E previously observed to correlate with sub endothelial pericapillary deposits in organ transplant rejection, and with clinical SLE flares. Conversely, significant C3 deposits on E were only observed among » of the patients. The decrease of CR1/E density, deposits of C4 fragments on E and previously reported detection of virus spikes or C3 on E among COVID-19 patients, suggest that the handling and clearance of immune complex or complement fragment coated cell debris may play an important role in the pathophysiology of SARS-CoV-2. Measurement of C4d deposits on E might represent a surrogate marker for assessing inflammation and complement activation occurring in organ capillaries and CR1/E decrease might represent a cumulative index of complement activation in COVID-19 patients. Taken together, these original findings highlight the participation of complement regulatory proteins and indicate that E are important in immune pathophysiology of COVID-19 patients. Besides a potential role for monitoring the course of disease, these observations suggest that novel therapies such as the use of CR1, or CR1-like molecules, in order to down regulate complement activation and inflammation, should be considered.