key: cord-0890155-zbhmtpyh authors: Patterson, B. K.; Seethamraju, H.; Dhody, K.; Corley, M. J.; Kazempour, K.; Lalezari, J. P.; Pang, A. P.; Sugai, C.; Francisco, E. B.; Pise, A.; Rodrigues, H.; Ryou, M.; Wu, H. L.; Webb, G. M.; Park, B. S.; Kelly, S.; Pourhassan, N.; Lelic, A.; Kdouh, L.; Herrera, M.; Hall, E.; Aklin, E.; Ndhlovu, L.; Sacha, J. B. title: Disruption of the CCL5/RANTES-CCR5 Pathway Restores Immune Homeostasis and Reduces Plasma Viral Load in Critical COVID-19 date: 2020-05-05 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.05.02.20084673 sha: 342c3b497dc44d2230964f6a9c6e07e4d5ee8510 doc_id: 890155 cord_uid: zbhmtpyh Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is now pandemic with nearly three million cases reported to date. Although the majority of COVID-19 patients experience only mild or moderate symptoms, a subset will progress to severe disease with pneumonia and acute respiratory distress syndrome (ARDS) requiring mechanical ventilation. Emerging results indicate a dysregulated immune response characterized by runaway inflammation, including cytokine release syndrome (CRS), as the major driver of pathology in severe COVID-19. With no treatments currently approved for COVID-19, therapeutics to prevent or treat the excessive inflammation in severe disease caused by SARS-CoV-2 infection are urgently needed. Here, in 10 terminally-ill, critical COVID-19 patients we report profound elevation of plasma IL-6 and CCL5 (RANTES), decreased CD8+ T cell levels, and SARS-CoV-2 plasma viremia. Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia. Consistent with reduction of plasma IL-6, single-cell RNA-sequencing revealed declines in transcriptomic myeloid cell clusters expressing IL-6 and interferon-related genes. These results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19. IL-1b, IL-6, and IL-8 (Fig. 1a -c) compared to healthy controls. In comparison to patients 98 with mild or moderate COVID-19, only IL-6 was present at significantly higher levels in 99 critically-ill patients. Of note, plasma CCL5 levels in the ten critically ill patients were 100 markedly elevated over those in both healthy controls and mild or moderate COVID-19 101 patients (Fig. 1d) . High levels of CCL5 can cause acute renal failure and liver toxicity 17,18 , 102 both common findings in COVID-19 infection. Indeed, the critically ill patients presented 103 with varying degrees of kidney and liver injury, although many had also previously 104 received kidney transplants 15 (Table 1 and Supplementary Fig. 1) . 105 At study day zero, all ten critically ill patients received a subcutaneous 700mg injection of 107 leronlimab following baseline blood collection. Because defining features of severe 108 COVID-19 disease include plasma IL-6 and T cell lymphopenia 2,19 , and we observed 109 >100-fold increased CCL5 levels compared to normal controls (Fig. 1d) , we longitudinally 110 monitored these parameters for two weeks after leronlimab treatment. A reduction of 111 plasma IL-6 was observed as early as three days following leronlimab and returned to 112 healthy control levels by day 14 (Fig. 2a) . In contrast, more variable levels were observed 113 with IL-1b, IL-8, and CCL5 after leronlimab treatment ( Supplementary Fig. 2 ). Following 114 leronlimab administration, a marked restoration of CD8+ T cells (Fig. 2b) and a 115 normalization of the CD4+ and CD8+ T cell ratio in blood was observed (Fig. 2c) . These 116 immunological changes occurred concomitant with full leronlimab CCR5 receptor 117 occupancy on the surface of CCR5+ T cells and macrophages (Fig. 2d, 2e) . Low levels 118 of SARS-CoV-2 have been detected, but not yet quantified in the plasma of COVID-19 119 patients 19 . We used high sensitivity, digital droplet PCR to quantify plasma SARS-CoV-2 120 All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. . https://doi.org/10.1101/2020.05.02.20084673 doi: medRxiv preprint Finally, to establish an unbiased gene repertoire for these COVID-19 patients, we 126 performed 10X Genomics 5' single cell RNA-sequencing of peripheral blood mononuclear 127 cells to evaluate transcriptional changes between an uninfected healthy donor and two of 128 the severe COVID-19 patients (P2 and P4) for which sufficient baseline, pre-leronlimab 129 treatment COVID-19 samples were available for this analysis. We identified 2,890 130 differentially expressed transcripts between the two groups and found that the two severe 131 , interferon (IFN)-, and chemokine-related genes compared to a healthy control (FDR < 133 0.05) (Supplementary Table 2 ). Notable genes overexpressed in COVID-19 samples 134 included chemokines (CXCL8, CCL4, CCL3), inflammatory and immune activation genes 135 (IL-1b, CD69), and the IFN-related genes (IFI27, IFITM3) ( Supplementary Fig. 3 ). We 136 also observed a downregulation of the effector molecule granzyme A and the 137 immunoregulatory gene KLRB1 compared to the healthy control. 138 To identify markers that would inform effective leronlimab treatment we conducted 140 differential expression analysis for the same two severe COVID-19 participants (P2 and 141 P4) for which baseline and day seven post leronlimab samples were available. Our 142 longitudinal COVID-19 single cell dataset profiled an estimated 4,105 cells at baseline 143 All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Here, we report on the involvement of the CCL5-CCR5 pathway in COVID-19 and present 156 data from ten critically ill patients with severe COVID-19 demonstrating reduction of 157 inflammation, restoration of T cell lymphocytopenia, and reduced SARS-CoV-2 plasma 158 viremia following leronlimab-mediated CCR5 blockade. Recent studies have found that a 159 significant number of COVID-19 patients experience increased risks of strokes, blood 160 clots and other thromboembolic events 21 . Platelet activation, which leads to the initiation 161 of the coagulation cascade, can be triggered by chemokines including CCL5 22 , 162 suggesting that leronlimab treatment may be beneficial beyond its immunomodulatory 163 effects on inflammation and hemostasis in COVID-19 patients. 164 165 All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. followed by a 30 min. incubation in the dark at room temperature. Cells were washed 211 All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. Leronlimab. Dr. Lalezari is a principal investigator for CytoDyn Inc. through his company 337 All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. . All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which this version posted May 5, 2020. . https://doi.org/10.1101/2020.05.02.20084673 doi: medRxiv preprint Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to