key: cord-1052353-jtq2enhw
authors: Patterson, Bruce K.; Seethamraju, Harish; Dhody, Kush; Corley, Michael J.; Kazempour, Kazem; Lalezari, Jay; Pang, Alina P.S.; Sugai, Christopher; Mahyari, Eisa; Francisco, Edgar B.; Pise, Amruta; Rodrigues, Hallison; Wu, Helen L.; Webb, Gabriela M.; Park, Byung S.; Kelly, Scott; Pourhassan, Nader; Lelic, Alina; Kdouh, Lama; Herrera, Monica; Hall, Eric; Bimber, Benjamin N.; Plassmeyer, Matthew; Gupta, Raavi; Alpan, Oral; O’Halloran, Jane A.; Mudd, Philip A.; Akalin, Enver; Ndhlovu, Lishomwa C.; Sacha, Jonah B.
title: CCR5 Inhibition in Critical COVID-19 Patients Decreases Inflammatory Cytokines, Increases CD8 T-Cells, and Decreases SARS-CoV2 RNA in Plasma by Day 14
date: 2020-11-10
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.10.101
sha: 8b6816a76fb36ed024f75708c2b11db6a71fa054
doc_id: 1052353
cord_uid: jtq2enhw

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 patients with severe COVID-19 disease. METHODS: In March 2020, ten terminally-ill, critical COVID-19 patients received two doses of leronlimab via individual emergency use indication (EIND). 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 6/10 patients survived, 2 extubated, and 1 patient was discharged. We observed complete CCR5 receptor occupancy in all donors by day 7. Compared to baseline, we observed a concomitant statistically significant reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and resolution of SARS-CoV2 plasma viremia (pVL) compared to controls. Further, the increase in CD8% was inversely correlated with reduction in pVL (r = −0.77, p = 0.0013). CONCLUSIONS: While the current study design precludes clinical efficacy inferences, these results implicate CCR5 as a therapeutic target for COVID-19 and form the basis for ongoing randomized clinical trials.

Since the initial cases of COVID-19 were reported from Wuhan, China in December 2019 (Huang et al., 2020) , SARS-CoV-2 has emerged as a global pandemic with an ever increasing number of severe and critical cases requiring invasive external ventilation that threatens to overwhelm health care systems (World Health Organization, 2020. While it remains unclear why COVID-19 patients experience a spectrum of clinical outcomes ranging from asymptomatic to severe disease, features of critical COVID-19 include rampant inflammation and cytokine release syndrome (CRS) leading to ARDS (Mehta et al., 2020 . Indeed, excessive immune cell infiltration into the lung, CRS, and ARDS have previously been described as defining features of severe disease in humans infected with the closely related betacoronaviruses SARS-CoV and MERS-CoV (Channappanavar and Perlman, 2017, Nicholls et al., 2003) . Because SARS-CoV-infected airway epithelial cells and macrophages express high levels of CCL5 (Law et al., 2005 , Yen et al., 2006 , a chemotactic molecule able to amplify inflammatory responses towards immunopathology, we hypothesized that disrupting the CCL5-CCR5 axis via leronlimab-mediated CCR5 blockade might prevent pulmonary trafficking of proinflammatory leukocytes and dampen pathogenic immune activation in Leronlimab, formerly PRO 140, is a CCR5-specific human IgG4 monoclonal antibody in development for HIV therapy as a once-weekly, at-home subcutaneous injection. In five completed and four ongoing HIV clinical trials where over 800 individuals have received leronlimab, no drug related deaths, serious injection site reactions, or drug-drug interactions were reported (Jacobson et al., 2008 , Jacobson et al., 2010a , Jacobson et at., 2010b , Dhody et al., 2018 . Subcutaneous, self-administration of leronlimab by patients facilitates simple, onceweekly dosing. In contrast to the small molecule CCR5 inhibitors that prevent HIV Env binding to CCR5 via allosteric modulation, leronlimab binds to the CCR5 extracellular loop 2 domain and Nterminus, thereby directly blocking the binding of HIV Env to the CCR5 co-receptor via a competitive mechanism. Leronlimab does not downregulate CCR5 surface expression or deplete J o u r n a l P r e -p r o o f CCR5-expressing cells, but does prevent CCL5-induced calcium mobilization in CCR5+ cells with an IC50 of 45 µg/ml (Olson et al., 1999) . This ability to specifically prevent CCL5-induced activation and chemotaxis of inflammatory CCR5+ macrophages and T cells suggests that leronlimab might be effective in mitigating pathologies involving the CCR5-ligand pathway.

All leronlimab-treated patients were enrolled in this study under an individual patient emergency use investigation new drug (EIND) via FDA emergency use authorization (EUA). The Albert Einstein College of Medicine Institution Review Board (IRB) reviewed and approved this study.

One 8 mL EDTA tube and one 4 mL plasma preparation (PPT) tube were drawn by venipuncture at Day 0 (pre-treatment), Day 3, Day 7, Day 14 post-treatment. Peripheral blood mononuclear cells were isolated from peripheral blood using Lymphoprep density gradient (STEMCELL Technologies, Vancouver, Canada). Aliquots of cells were frozen in media that contained90% fetal bovine serum (Hyclone, Logan, UT) and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and stored at -70C. The five COVID observational control patients are part of a prospective observational cohort of subjects with viral respiratory illness symptoms who presented to Barnes Jewish Hospital, St. Louis Children's Hospital or affiliated Barnes Jewish Hospital testing sites located in Saint Louis, Missouri, USA. Inclusion criteria required that subjects were symptomatic and had a physician-ordered SARS-CoV-2 test performed in the course of their normal clinical care. All samples were collected at the time of enrollment, which was during or immediately following evaluation in a medical facility. The study was reviewed and approved by the Washington University in Saint Louis Institutional Review Board (WU-350 study approval # 202003085). The study complied with the ethical standards of the Helsinki Declaration.

Assessment of plasma cytokine and chemokine levels Fresh plasma was used for cytokine quantification using a customized 13-plex bead-based 

The QIAamp Viral Mini Kit (Qiagen, Catalog #52906) was used to extract nucleic acids from 300-400 µL from plasma sample according to instructions from the manufacturer and eluted in 50 µL of AVE buffer (RNase-free water with 0.04% sodium azide). The purified nucleic acids were used immediately with the Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad, Hercules, CA). The panel is designed for specific detection of the 2019-nCoV (two primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RP) in control samples and clinical specimens was used. RNA isolated and purified from the plasma samples (5.5 µL) were added to the mastermix comprised of 1.1 µL of 2019-nCoV triplex assay, 2.2 µL of reverse transcriptase, 5.5 µL of supermix, 1.1 µL of Dithiothreitol (DTT) and 6.6 µL of nuclease-free water.

The mixtures were then fractionated into up to 20,000 nanoliter-sized droplets in the form of a water-in-oil emulsion in the QX200 Automated Droplet Generator (Bio-Rad, Hercules CA). The 96well RT-ddPCR ready plate containing droplets was sealed with foil using a plate sealer and thermocycled to achieve reverse transcription of RNA followed by PCR amplification of J o u r n a l P r e -p r o o f cDNA in a C1000 Touch thermocycler (Bio-Rad, Hercules CA). Subsequent to PCR, the plate was loaded into the QX200 Droplet Reader (Bio-Rad, Hercules CA) and the fluorescence intensity of each droplet was measured in two channels (FAM and HEX). The fluorescence data is then analyzed by the QuantaSoft 1.7 and QuantaSoft Analysis Pro 1.0 Software to determine the presence of SARS-CoV-2 N1 and N2 in the specimen. The limit of detection was determined as shown in Supplementary Figure 1 .

Temperature ( 

Ten critical COVID-19 patients at the Montefiore Medical Center received leronlimab via FDA-J o u r n a l P r e -p r o o f approved emergency investigational new drug (EIND) requests for individual patient use (Table   1 ). These confirmed SARS-CoV-2 positive patients had significant pre-existing co-morbidities and were receiving intensive care treatment including mechanical ventilation or supplemental oxygen for ARDS. Consistent with previous reports of severe COVID-19 disease (Huang et al., 2020) , these patients showed evidence of lymphopenia with liver and kidney damage ( Supplementary Fig. 2 ) (Akalin et al., 2020) . Four of the patients died during the fourteen-day study period due to a combination of disease complications and severe constraints on medical equipment culminating in medical triage. Although this EIND study lacks a placebo control group for comparison, a recent study of other critically ill COVID-19 patients in the New York City area indicates mortality rates as high as 88% (Richarson et al., 2020).

Hyper immune activation and CRS are present in cases of severe COVID-19 (Mehta et al., 2020) . Indeed, at leronlimab treatment baseline, signatures of CRS were present in the plasma of all Ten patients in the form of significantly elevated levels of the inflammatory cytokines IL-1β, IL-6, and IL-8 ( Fig. 1A -C) compared to healthy controls. In comparison to patients with mild or moderate COVID-19, only IL-6 was present at significantly higher levels in the critically-ill patients. Of note, plasma CCL5 levels in the ten critically ill patients were markedly elevated over those in both healthy controls and mild or moderate COVID-19 patients (Fig. 1D ). High levels of CCL5 can cause acute renal failure and liver toxicity (Yu et al., 2016 , Chen et al., 2020 , both common findings in COVID-19 infection. Indeed, the critically ill patients presented with varying degrees of kidney and liver injury, although many had also previously received kidney transplants (Akalin et al., 2020) (Table 1 and Supplementary Fig. 2 ). Low levels of SARS-CoV-2 have been detected, but not yet quantified in the plasma of COVID-19 patients (Lescure et al., 2020) . We used high sensitivity, digital droplet PCR to quantify plasma SARS-CoV-2 viremia at baseline. SARS-CoV-2 was found in the plasma of all ten critically ill patients, in contrast to no viremia in healthy controls and in only one patient with mild/moderate COVID-19, further underscoring the critical nature of COVID-19 in these patients (Fig. 1E ).

At study day zero, all ten critically ill patients received a subcutaneous 700mg injection of leronlimab following baseline blood collection. Patients subsequently received a second subcutaneous injection of 700mg Leronlimab at study day 7. Because defining features of severe COVID-19 disease include plasma IL-6 and T cell lymphopenia (Huang et al., 2020 , Lescure et al., 2020 , we longitudinally monitored these parameters for two weeks after the first leronlimab treatment. A reduction of plasma IL-6 was observed as early as three days following leronlimab and returned to healthy control levels by day 14 (Fig. 2A) . In contrast to IL-6, other cytokine and chemokine levels were more variable after leronlimab treatment ( Supplementary Fig. 3 ). Following leronlimab administration, a marked restoration of CD8+ T cells (Fig. 2B ) and a normalization of the CD4+ and CD8+ T cell ratio in blood was observed (Fig. 2C) . These immunological changes occurred concomitant with leronlimab CCR5 receptor occupancy on the surface of CCR5+ T cells and monocytes (Fig. 2D, 2E) . Table 2 ). The top pathways enriched in this set included multiple TCR signaling pathways, which included the genes LCK, CD3E and NFKBIA (Figure 3 ). Because differences in cellularity between donors will confound global gene expression analyses, we extracted the myeloid cell cluster and performed differential expression analyses using only these cells. We To identify markers that would inform effective leronlimab treatment, we next compared gene expression within the two severe COVID-19 participants, contrasting baseline (D0) and day seven post leronlimab (D7). When performing bulk DE on total PBMC, we identified 14 DE genes, though as above this will be confounded by the differences in cell composition. It should be noted that this set included a significant drop in CD44, a downstream target of IL-6 signaling (Vincent and Mechti, 2004) , and a number of genes involved in neutrophil degranulation (ITGB2, EEF2, HSP90AA1, and ALDOA). Next, we performed DE specifically on myeloid cells, identifying 73 DE transcripts in this subset (Wilcox, adj. p.val< 0.1; Supplementary Table 2 ). In line with the decrease of IL-6 protein levels observed in plasma, we observed a significant decrease in expression of many IL-6 responsive genes, including TGFB1, IFI30, and LY6E, consistent with reports of monocyte/macrophages repolarization following CCR5 blockade (Halama et al., 2016) . We also observed that myeloid cells expressing chemokine and IFN-J o u r n a l P r e -p r o o f related genes such as IFNGR2, IFITM2, TALDO1, and were significantly downregulated at day 7 post leronlimab compared to baseline (Supplementary Table 2 ). These transcriptomic findings further underscore the potential impact of Leronlimab-mediated CCR5 blockade on the inflammatory state in COVID-19.

Here, we report on the involvement of the chemokine receptor CCR5 in COVID-19 and present data from ten critically ill patients with severe COVID-19 demonstrating reduction of inflammation, restoration of T cell lymphocytopenia, and reduced SARS-CoV-2 plasma viremia following leronlimab-mediated CCR5 blockade. We found statistically significant elevation of IL-1β, IL-6, IL-8, and RANTES in these critically ill patients compared to healthy controls.

Elevations of these cytokines has been the hallmark of COVID-19 and elevation of the chemokine CCL5/RANTES has been demonstrated across the COVID-19 disease spectrum from mild to severe patients. RANTES was elevated to a greater extent than the other CCR5 binding chemokines MIP-1α and MIP-1β most likely due to the production of RANTES in respiratory epithelial cells (in addition to immune cells) in respiratory viral infections (Schroth MK et al., 1999) . We also show profound reduction of CD8% with concomitant increases in CD4/CD8 ratios. In this cohort of critical COVID-19 patients, SARS-CoV-2 RNA was detectable and quantifiable in plasma samples from all patients. For the first time, we demonstrate that restoration of the CD8 T-cell numbers correlates with decreases in plasma viral load with statistical significance. These data in sum show that therapy with a CCR5 antagonist reduces the cytokine storm, resolves the profound CD-8 T-cell lymphopenia, and reduces plasma viral load to undetectable levels by Day 14. These data corroborate more recent data suggesting the potential for targeting CCR5 as a therapeutic in COVID-19 (Chua et al., 2020) .

Recent studies have found that a significant number of COVID-19 patients experience increased risks of strokes, blood clots and other thromboembolic events (Grillet et al., 2020) . Platelet 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CCL5 (D) , and SARS-CoV-2 RNA copies (E) in patients with mild/moderate (purple symbols, n=8) and critical (red symbols, n=10 panels a-d or n=7 panel e) COVID-19 disease, compared to healthy controls (black symbols, n=10). Dashed line indicated limit of detection. Graphs display p-values calculated by Dunn's Kruskal-Wallis test: *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Graph displays p-value calculated by Mann-Whitney test: *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (G) Plot showing %CD8 in blood and SARS-CoV-2 plasma viral load in 7 critically ill COVID-19 patients at days 0, 7, and 14 post-leronlimab (n=20). Graph displays rho (ρ) and p-value calculated by repeated measures correlation: *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. 95% confidence interval for the repeated measures correlation was -0.93 to 0.35. 

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We gratefully acknowledge the patient participants and their caregivers Kristine Stryker, Victoria 

J o u r n a l P r e -p r o o f = vancomycin, CAX = ceftriaxone, LPV/RTV = lopinavir/ritonavir, TZP = piperacillintazobactam, CEF = cefepime, NC = nasal canula, NRB = non-rebreather mask, TBD = to be determined, *Patient declined intubation due to poor baseline pulmonary status