key: cord-0714897-kl3fvlxw
authors: Cross, Robert W.; Prasad, Abhishek N.; Borisevich, Viktoriya; Woolsey, Courtney; Agans, Krystle N.; Deer, Daniel J.; Dobias, Natalie S.; Geisbert, Joan B.; Fenton, Karla A.; Geisbert, Thomas W.
title: Use of convalescent serum reduces severity of COVID-19 in nonhuman primates
date: 2021-02-23
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.108837
sha: 17a292f965a2ac5ea9c2f06b8b96750586f29900
doc_id: 714897
cord_uid: kl3fvlxw

Passive transfer of convalescent plasma or serum is a time-honored strategy for treating infectious diseases. Human convalescent plasma containing antibodies against SARS-CoV-2 is currently being used to treat COVID-19 patients where clinical efficacy trials are ongoing. Here, we assess therapeutic passive transfer in groups of SARS-CoV-2-infected African green monkeys with convalescent sera containing either high or low anti-SARS-CoV-2 neutralizing antibody titers. Differences in viral load and pathology are minimal between monkeys that receive the lower titer convalescent sera and untreated controls. However, lower levels of SARS-CoV-2 in respiratory compartments, reduced severity of virus-associated lung pathology, and reductions in coagulopathy and inflammatory processes are observed in monkeys that receive high titer sera versus untreated controls. Our data indicate convalescent plasma therapy in humans may be an effective strategy provided donor sera contain high anti-SARS-CoV-2 neutralizing titers given in early stages of disease.

binding and neutralizing potential of the treatment in vivo, sera from the convalescent sera-92 treated AGMs was fractionated from whole blood collected 2 and 5 dpi and assessed for binding 93 by ELISA and neutralizing activity by PRNT 50 (Figure 1a,b) . The binding titer was 1:51,200 94 (total virus), 1:25,600 (anti-Nucleoprotein), and 1:12,800 (anti-spike RBD); which corresponded 95 to a PRNT 50 in pooled sera of ~ 1:2048 for this cohort (designated "high dose"; "HD"). The 96 J o u r n a l P r e -p r o o f second experimental cohort received pooled sera from a group of three AGMs euthanized at the 97 scheduled study endpoint 34 days after challenge with SARS-CoV-2 (Cross et al., 2020); the 98 binding titer was 1:3,200 (total virus, anti-Nucleoprotein, and anti-spike RBD); which 99 corresponded to a PRNT 50 for pooled sera of ~ 1:128 (designated as "low dose"; "LD").

Animals were longitudinally monitored for clinical signs of illness and euthanized 5 dpi. showed mild clinical illness (Table 1) . Shifts in leukocyte populations as compared to pre-106 challenge baseline counts; specifically, lymphocytopenia, generalized granulocytopenia 107 (neutropenia, eosinopenia, and/or basopenia), and mild to moderate thrombocytopenia were 108 common to most animals regardless of cohort starting approximately 2 dpi (Table 1) . Four 109 animals (HD-AGM-3, LD-AGM-3, LD-AGM-4, and C-AGM-2) experienced monocytosis 110 beginning 2-4 dpi; in two of these animals (HD-AGM-3 and LD-AGM-4) this coincided with 111 generalized granulocytosis (neutrophilia, eosinophilia, and/or basophilia). Prothrombin time (PT) 112 was largely unaffected, yet a significantly prolonged coagulation time was noted at 4 dpi for 113 activated partial thromboplastin time (aPTT) in HD versus control group (p=0.0459; two-way 114 ANOVA with Tukey's multiple comparison); decreases in levels of thrombocytes were more 115 notable in control animals; and increases in circulating fibrinogen were generally more 116 pronounced in the control group compared to either of the experimental groups ( Figure S1 ). 117 These results suggest that while all animals appear to have experienced varying degrees of 118 coagulopathy, treatment with sera with a higher SARS-CoV-2 neutralizing capacity may have 119 J o u r n a l P r e -p r o o f partially ameliorated disease in the HD group. However, differences in these parameters were 120 not statistically significant for most time points due to the small cohort sizes and individual 121 animal variability. Serum markers of renal and/or hepatic function (CRE, ALT, AST) were 122 mildly elevated in most animals from treatment groups as well as the control group, while CRP, 123 a marker of acute systemic inflammation, was mild to moderately (1-10 fold over baseline) 124 elevated 4-5 dpi in all but a single animal (LD-AGM-2). Circulating SARS-CoV-2 specific antibodies to total virus, nucleoprotein, and the receptor 128 binding domain (RBD) of the spike protein were higher in all animals on 2 dpi of the HD group 129 compared to the LD group and untreated subjects, but by day 5, spike RBD titers in 2/4 HD 130 animals began to decline slightly (Figure 2a,b,c) . Three of four animals in the HD-treated group Assessment of viral load 144 We next assessed viral load in whole blood and mucosal swabs on 0, 2, 3, 4, and 5 dpi, were observed in all of the HD-treated animals as compared to control groups beginning 2 dpi 229 and trending through the remainder of the study (Figure 6g, h, i, j, and k) . The data sets used and/or analyzed during the current study are available from the corresponding 441 author on reasonable request.

Data and software availability 443 The datasets supporting the current study have not been deposited in a public repository because 444 an appropriate repository for the datasets is not available. Datasets are available from the 445 corresponding author on reasonable request. This study did not generate code. 

On specified procedure days (days 0, 2, 3, 4, 5), 100 µl of blood was added to 600 µl of AVL Samples were processed in duplicate following the kit instructions and recommendations.

Following bead staining and washing, 4000 bead events were collected on a FACS Canto II 594 cytometer (BD Biosciences) using BD FACS Diva software. The raw .fcs files were analyzed 595 with BioLegend's cloud-based LEGENDplex™ Data Analysis Software.

The data was analyzed and graphed in GraphPad Prism 9.0.0. Lesion severity scores and tissue 598 PCR and plaque assay titers were analyzed using either a one-way ANOVA or a two-way 599 ANOVA supported by Tukey's multiple comparisons test. ELISAs and LegendPlex assays were h.

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. Subject survived to study endpoint.Monocytosis (d4, 5); neutrophilia (d4); eosinophilia (d4); basophilia (d4); mild thrombocytopenia (d4, 5); ↑ ALT (d4); ↑ AST (d3, 4); ↑↑ CRP (d4) C-AGM-1 M None. Subject survived to study endpoint (d5). Neutrophilia (d4); eosinophilia (d4); basophilia (d4); mild thrombocytopenia (d5); ↑ ALT (d3); ↑ AST (d3, 5); ↑ CRP (d4, 5) C-AGM-2 M None. Subject survived to study endpoint (d5). Monocytosis (d2, 4); neutropenia (d4); eosinopenia (d2, 4); basopenia (d2-5); thrombocytopenia (d3); mild thrombocytopenia (d4); ↑ CRE (d4); ↑ AST (d3-5); ↑ CRP (d4, 5)Days after SARS-CoV-2 challenge are in parentheses/brackets. All reported findings are in comparison to baseline (d0) values. Decreased appetite is defined as some food but not all food consumed from the previous day. Anorexia is defined as no food consumed from the previous day. Lymphocytopenia, monocytopenia, erythrocytopenia, thrombocytopenia, neutropenia, eosinopenia, and basopenia are defined by a ≥35% drop in numbers of lymphocytes, monocytes, erythrocytes, platelets, neutrophils, eosinophils, and basophils, respectively. Thrombocytopenia is defined as mild if the drop in number of platelets is 25%-34.9%. Lymphocytosis, monocytosis, neutrophilia, eosinophilia, and basophilia are defined by a 100% or greater increase in numbers of lymphocytes, monocytes, neutrophils, eosinophils, or basophils, respectively. Hyperglycemia is defined as a 100% or greater increase in levels of glucose. Hypoglycemia is defined by a ≥25% decrease in levels of glucose. Hypoalbuminemia is defined by a ≥25% decrease in levels of albumin. Hypoproteinemia is defined by a ≥25% decrease in levels of total protein. Hypoamylasemia is defined by a ≥25% decrease in levels of serum amylase. Hypocalcemia is defined by a ≥25% decrease in levels of serum calcium. Hypercapnia was defined as having a partial CO2 >4 mmHg over d0 baseline values. Increases in ALT, AST, ALP, CRE, CRP, Hct, and Hgb were graded on the following scale: