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.

Genome-wide DNA methylation profiling of peripheral blood reveals an epigenetic signature associated with severe COVID-19.

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic RNA virus causing coronavirus disease 2019 (COVID-19) in humans. Although most patients with COVID-19 have mild illness and may be asymptomatic, some will develop severe pneumonia, acute respiratory distress syndrome, multi-organ failure, and death. RNA viruses such as SARS-CoV-2 are capable of hijacking the epigenetic landscape of host immune cells to evade antiviral defense. Yet, there remain considerable gaps in our understanding of immune cell epigenetic changes associated with severe SARS-CoV-2 infection pathology. Here, we examined genome-wide DNA methylation (DNAm) profiles of peripheral blood mononuclear cells from 9 terminally-ill, critical COVID-19 patients with confirmed SARS-CoV-2 plasma viremia compared with uninfected, hospitalized influenza, untreated primary HIV infection, and mild/moderate COVID-19 HIV coinfected individuals. Cell-type deconvolution analyses confirmed lymphopenia in severe COVID-19 and revealed a high percentage of estimated neutrophils suggesting perturbations to DNAm associated with granulopoiesis. We observed a distinct DNAm signature of severe COVID-19 characterized by hypermethylation of IFN-related genes and hypomethylation of inflammatory genes, reinforcing observations in infection models and single-cell transcriptional studies of severe COVID-19. Epigenetic clock analyses revealed severe COVID-19 was associated with an increased DNAm age and elevated mortality risk according to GrimAge, further validating the epigenetic clock as a predictor of disease and mortality risk. Our epigenetic results reveal a discovery DNAm signature of severe COVID-19 in blood potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against SARS-CoV-2.

Immune-Based Prediction of COVID-19 Severity and Chronicity Decoded Using Machine Learning.

Expression of CCR5 and its cognate ligands have been implicated in COVID-19 pathogenesis, consequently therapeutics directed against CCR5 are being investigated. Here, we explored the role of CCR5 and its ligands across the immunologic spectrum of COVID-19. We used a bioinformatics approach to predict and model the immunologic phases of COVID so that effective treatment strategies can be devised and monitored. We investigated 224 individuals including healthy controls and patients spanning the COVID-19 disease continuum. We assessed the plasma and isolated peripheral blood mononuclear cells (PBMCs) from 29 healthy controls, 26 Mild-Moderate COVID-19 individuals, 48 Severe COVID-19 individuals, and 121 individuals with post-acute sequelae of COVID-19 (PASC) symptoms. Immune subset profiling and a 14-plex cytokine panel were run on all patients from each group. B-cells were significantly elevated compared to healthy control individuals (P<0.001) as was the CD14+, CD16+, CCR5+ monocytic subset (P<0.001). CD4 and CD8 positive T-cells expressing PD-1 as well as T-regulatory cells were significantly lower than healthy controls (P<0.001 and P=0.01 respectively). CCL5/RANTES, IL-2, IL-4, CCL3, IL-6, IL-10, IFN-γ, and VEGF were all significantly elevated compared to healthy controls (all P<0.001). Conversely GM-CSF and CCL4 were in significantly lower levels than healthy controls (P=0.01). Data were further analyzed and the classes were balanced using SMOTE. With a balanced working dataset, we constructed 3 random forest classifiers: a multi-class predictor, a Severe disease group binary classifier and a PASC binary classifier. Models were also analyzed for feature importance to identify relevant cytokines to generate a disease score. Multi-class models generated a score specific for the PASC patients and defined as S1 = (IFN-γ + IL-2)/CCL4-MIP-1ß. Second, a score for the Severe COVID-19 patients was defined as S2 = (IL-6+sCD40L/1000 + VEGF/10 + 10*IL-10)/(IL-2 + IL-8). Severe COVID-19 patients are characterized by excessive inflammation and dysregulated T cell activation, recruitment, and counteracting activities. While PASC patients are characterized by a profile able to induce the activation of effector T cells with pro-inflammatory properties and the capacity of generating an effective immune response to eliminate the virus but without the proper recruitment signals to attract activated T cells.

CCR5 inhibition in critical COVID-19 patients decreases inflammatory cytokines, increases CD8 T-cells, and decreases SARS-CoV2 RNA in plasma by day 14.

OBJECTIVE: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now a global pandemic. Emerging results indicate a dysregulated immune response. Given the role of CCR5 in immune cell migration and inflammation, we investigated the impact of CCR5 blockade via the CCR5-specific antibody leronlimab on clinical, immunological, and virological parameters in severe COVID-19 patients. METHODS: In March 2020, 10 terminally ill, critical COVID-19 patients received two doses of leronlimab via individual emergency use indication. We analyzed changes in clinical presentation, immune cell populations, inflammation, as well as SARS-CoV-2 plasma viremia before and 14 days after treatment. RESULTS: Over the 14-day study period, six patients survived, two were extubated, and one discharged. We observed complete CCR5 receptor occupancy in all donors by day 7. Compared with the baseline, we observed a concomitant statistically significant reduction in plasma IL-6, restoration of the CD4/CD8 ratio, and resolution of SARS-CoV2 plasma viremia (pVL). Furthermore, the increase in the CD8 percentage was inversely correlated with the reduction in pVL (r = -0.77, p = 0.0013). CONCLUSIONS: Our study design precludes clinical efficacy inferences but the results implicate CCR5 as a therapeutic target for COVID-19 and they form the basis for ongoing randomized clinical trials.

SARS-CoV-2 Viral RNA Shedding for More Than 87 Days in an Individual With an Impaired CD8+ T Cell Response.

Prolonged shedding of viral RNA occurs in some individuals following SARS-CoV-2 infection. We perform comprehensive immunologic evaluation of one individual with prolonged shedding. The case subject recovered from severe COVID-19 and tested positive for SARS-CoV-2 viral RNA repeatedly as many as 87 days after the first positive test, 97 days after symptom onset. The subject did not have any associated rise in anti-Spike protein antibody titers or plasma neutralization activity, arguing against re-infection. This index subject exhibited a profoundly diminished circulating CD8+ T cell population and correspondingly low SARS-CoV-2-specific CD8+ T cell responses when compared with a cohort of other recovering COVID-19 subjects. CD4+ T cell responses and neutralizing antibody responses developed as expected in this individual. Our results demonstrate that detectable viral RNA shedding in the upper airway can occur more than 3 months following infection in some individuals with COVID-19 and suggest that impaired CD8+ T cells may play a role in prolonged viral RNA shedding.

Disruption of CCR5 signaling to treat COVID-19-associated cytokine storm: Case series of four critically ill patients treated with leronlimab.

Coronavirus disease 2019 (COVID-19) is associated with considerable morbidity and mortality. The number of confirmed cases of infection with SARS-CoV-2, the virus causing COVID-19 continues to escalate with over 70 million confirmed cases and over 1.6 million confirmed deaths. Severe-to-critical COVID-19 is associated with a dysregulated host immune response to the virus, which is thought to lead to pathogenic immune dysregulation and end-organ damage. Presently few effective treatment options are available to treat COVID-19. Leronlimab is a humanized IgG4, kappa monoclonal antibody that blocks C-C chemokine receptor type 5 (CCR5). It has been shown that in patients with severe COVID-19 treatment with leronlimab reduces elevated plasma IL-6 and chemokine ligand 5 (CCL5), and normalized CD4/CD8 ratios. We administered leronlimab to 4 critically ill COVID-19 patients in intensive care. All 4 of these patients improved clinically as measured by vasopressor support, and discontinuation of hemodialysis and mechanical ventilation. Following administration of leronlimab there was a statistically significant decrease in IL-6 observed in patient A (p=0.034) from day 0-7 and patient D (p=0.027) from day 0-14. This corresponds to restoration of the immune function as measured by CD4+/CD8+ T cell ratio. Although two of the patients went on to survive the other two subsequently died of surgical complications after an initial recovery from SARS-CoV-2 infection.