key: cord-339128-npfoircv
authors: Blair, Robert V.; Vaccari, Monica; Doyle-Meyers, Lara A.; Roy, Chad J.; Russell-Lodrigue, Kasi; Fahlberg, Marissa; Monjure, Chris J.; Beddingfield, Brandon; Plante, Kenneth S.; Plante, Jessica A.; Weaver, Scott C.; Qin, Xuebin; Midkiff, Cecily C.; Lehmicke, Gabrielle; Golden, Nadia; Threeton, Breanna; Penney, Toni; Allers, Carolina; Barnes, Mary B.; Pattison, Melissa; Datta, Prasun K.; Maness, Nicholas J.; Birnbaum, Angela; Fischer, Tracy; Bohm, Rudolf P.; Rappaport, Jay
title: Acute Respiratory Distress in Aged, SARS-CoV-2 Infected African Green Monkeys but not Rhesus Macaques
date: 2020-11-07
journal: Am J Pathol
DOI: 10.1016/j.ajpath.2020.10.016
sha: 
doc_id: 339128
cord_uid: npfoircv

SARS-CoV-2 induces a wide range of disease severity ranging from asymptomatic infection, to a life-threating illness, particularly in the elderly and persons with comorbid conditions. Among those persons with serious COVID-19 disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19 induced ARDS and evaluate therapeutic strategies. Here we report ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that demonstrated pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. We found dramatic increases in circulating cytokines in three of four infected, aged AGMs but not in infected RMs. All of the AGMs showed increased levels of plasma IL-6 compared to baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2 infection. Together, our results show that both RM and AGM are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations including ARDS.

SARS-CoV-2 induces a wide range of disease severity ranging from asymptomatic infection, to a life-threating illness, particularly in the elderly and persons with comorbid conditions. Among those persons with serious COVID-19 disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19 induced ARDS and evaluate therapeutic strategies. Here we report ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that demonstrated pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. We found dramatic increases in circulating cytokines in three of four infected, aged AGMs but not in infected RMs.

Infection with SARS-CoV-2 and the development of COVID-19 is accompanied by a mild respiratory disease for most individuals. However, a small subset progress to develop severe respiratory disease which, in some cases, is fatal 1 . The most severely affected individuals often present with a fever, cough, dyspnea, and bilateral radiographic opacities that, in the majority of critically ill patients, progresses to acute respiratory distress syndrome (ARDS) 2 . The onset of ARDS is often associated with an increase in circulating pro-inflammatory cytokines often referred to as a "cytokine storm" 3, 4 . Worsening of disease can be seen in the context of declining viral loads and markedly elevated cytokines suggesting a role for these inflammatory responses in disease progression and immunopathology 5 . Research into the causes and mechanisms of the most severe manifestations of COVID-19 is needed to facilitate the development of prophylactic and therapeutic approaches that can prevent this life-threatening outcome.

Nonhuman primates (NHPs) are ideally suited to model respiratory human viral infections because of the similarities to human respiratory anatomy and immunologic responses when compared to other animal species. Several NHP species have been successfully employed to model pathogenesis 6-10 and test vaccine candidates [11] [12] [13] [14] for SARS-CoV-2. These prior studies have shown NHPs are susceptible to infection and develop mild to moderate disease, but none has been able to recapitulate the rapid clinical deterioration seen in people with severe disease and ARDS. Age is a well-established risk factor for severe disease and death in humans infected with SARS-CoV-2 2, 15, 16 ; therefore, older RM and AGMs were challenged with SARS-CoV-2 J o u r n a l P r e -p r o o f via two routes (aerosol and mulitroute) to see if a similar more severe disease phenotype was observed in aged cohorts after aerosol exposure.

This report describes the sudden and rapid health deterioration of two out of four aged AGMs experimentally infected with SARS-CoV-2. The two affected animals developed ARDS, and elevated circulating cytokines similar to the complications reported in 5-13% of COVID-19 patients 17 .

The Institutional Animal Care and Use Committee of Tulane University reviewed and approved all the procedures for this experiment. The Tulane National Primate Research Center is fully accredited by the AAALAC. All animals were cared for in accordance with the ILAR Guide for the Care and Use of Laboratory Animals 8 th Edition 18 J o u r n a l P r e -p r o o f Animals and procedures A total of eight animals, four aged (≈16 years of age), wild-caught AGM (2M, 2F) and four, adult (13-15 years of age) RM (3M, 1F) were used in this study. Animals (n=4) were exposed to SARS-CoV-2 either by small particle aerosol 20 or multiroute combination. The 4 animals (AGM1, AGM4, RM3, RM4) were exposed by aerosol and received an inhaled dose of approximately 2x10 3 TCID 50 . The other four animals (AGM2, AGM3, RM1, RM2) were exposed by inoculating a cumulative dose of 3.61x10 6 PFU through multiple routes (oral, 1 mL; nasal, 1mL; intratracheal, 1 mL; conjunctival, 50 µL per eye). Animals were observed for 28 days including twice daily monitoring. Pre-and postexposure samples included blood, CSF, feces, urine, bronchioalveolar lavage, and mucosal swabs (buccal, nasal, pharyngeal, rectal, vaginal, and bronchial brush). Blood was collected at postexposure days -14, 1, 3 (aerosol) or 4 (multiroute), 7, 14, 21, and at necropsy. CSF, feces, urine, bronchioalveolar lavage, and mucosal swabs were collected at post exposure days -14, 7, 14, 21, and at necropsy. Physical exam, plethysmography, and imaging (radiographs and PET/CT) occurred 7 days prior to exposure and then weekly thereafter. Animals were euthanized for necropsy after three weeks post exposure, or when humane end points were reached. Samples from the left anterior and caudal lung lobes were collected fresh and in media for further processing. All right lung lobes were infused and stored in fixative for microscopic evaluation. The remainder of the necropsy was performed routinely with collection of tissues in media, fixative, or fresh frozen.

Pulmonary pathology was scored using two separate random forest tissue segmentation algorithms trained by a veterinary pathologist in an unblinded fashion to recognize fibrin and J o u r n a l P r e -p r o o f edema and cellular inflammation using HALO software (Indica Labs, Albuquerque, NM). Tissue sections from each of the right lung lobes was segmented using the trained algorithms to quantify the percentage of tissue effected by fibrin and edema or cellular inflammation. The percentage of inflammation was converted to a pathology score based on the following scoring system: Fibrin and Edema score 0=0-2%, 1=2-5%, 2=5-15%, 3=15-30%, 4=>30% and Cellular Inflammation Score 0=0-0.5%, 1=0.5-3%, 2=3-6%, 3=6-12%, 4=>12%. The "Histopathology score" was made by summating the fibrin and edema and cellular inflammation scores for each lobe. The reaction master mix were added using an X-stream repeating pipette (Eppendorf, Hauppauge, NY) to the microtiter plates which were covered with optical film (cat. #4311971; Thermo Fisher), vortexed, and pulse centrifuged. The RT-qPCR reaction was subjected to RT-qPCR a program of, UNG incubation at 25°C for 2 minutes, RT incubation at 50°C for 15 minutes, and an enzyme activation at 95°C for 2 minutes followed by 40 cycles of a denaturing step at 95°C for 3 seconds and annealing at 60°C for 30 seconds. Heatmaps were generated using the 'pheatmap' package in R 21 . Data were normalized by dividing raw values at week 1 and necropsy by baseline values for each animal, followed by the application of log2. Values below the limit of detection were replaced with the lowest limit of detection value based on the standard curve for each run, or with the lowest value detected during the run, whichever was smaller. Polar coordinate plots were generated using the 'ggplot2' package in R 22 , using the same normalized data shown in the heatmap. Scatterplots were drawn using raw data points and display Pearson's correlation coefficients.

Serum samples collected at preinfection and weekly post-infection were tested for binding IgG antibodies against SARS-CoV-2 S1/S2 proteins using an ELISA kit from XpressBio (cat# 

Four, aged, AGMs and four RM, thirteen to fifteen years of age, were exposed by two routes to SARS-CoV-2 isolate USA-WA1/2020. Four animals (AGM1, AGM4, RM3, RM4) were exposed via small particle aerosol and four animals (AGM 2, AGM3, RM1, RM2) were exposed via multiple route installation (Table 1) . SARS-CoV-2 RNA was detectable in swabs obtained from mucosal sites in all eight animals ( Figure 1 ). The highest levels of viral RNA were detected in the pharynx and nasal cavity (Figure 1 , B and C). Rectal swabs contained high viral RNA loads similar to reports in humans ( Figure 1F Figure S1 ). The day prior (7-and 21 DPI) all animals underwent a complete physical evaluation and an extensive sample collection protocol including fluid, stool, swab, and bronchial brush collection, no remarkable findings were noted at that time in any of the animals.

In the 24 hours following sample collection (8 DPI for AGM1 and 22 DPI for AGM2) both animals developed mild tachypnea that progressed to severe respiratory distress that included dyspnea, tachypnea, hypothermia, and an SpO 2 of 77% (Supplemental Figure S1 ). No significant clinical findings were observed in any of the remaining animals after 22 DPI.

Thoracic radiographs for AGM1 and AGM2 revealed a diffuse alveolar pattern throughout the right lung fields and a lobar sign in the caudal dorsal lung field. In AGM2 the left caudal lung lobe also contained a mild alveolar pattern. These findings were in stark contrast to the radiographs from the day before highlighting the rapid disease progression (Figure 2, A and B ). RM1 had a focal pulmonary scar in the right caudal lung lobe surrounded by acute hemorrhage (Supplemental Figure S2E ). The lungs of the remaining animals (AGM4, RM2, RM3, and RM4) were grossly normal.

Histopathologic findings in the lungs of AGM1 and AGM2 were similar and characterized by alveoli that were filled with fibrin, hemorrhage, and proteinaceous fluid ( Figure 3A ). Alveoli were multifocally lined by hyaline membranes and/or type II pneumocytes, consistent with diffuse alveolar damage ( Figure 3B ). Alveoli contained rare multinucleated syncytial cells J o u r n a l P r e -p r o o f ( Figure 3C ). Fluorescent immunohistochemistry identified low numbers of SARS-CoV-2 infected cells in AGM1, but not AGM2. (Figure 3D ). The animals that survived to study endpoint exhibited minimal to mild interstitial inflammation (Supplemental Figure S3 and S4) .

Three out of four rhesus macaques (RM1, RM2, and RM4) had microscopic evidence of aspiration pneumonia characterized by foreign plant material within bronchioles. Histopathologic lesions in other tissues were mild and interpreted as not significant in all eight animals (Supplemental Table S1 ).

A group of cytokines similar to those observed in human COVID-19 was upregulated in the two animals that progressed to ARDS (AGM1 and AGM2) at the time of necropsy compared to baseline levels ( Figure 4A and Supplemental Figure S5 25 . Apart from the severe phenotype observed in two of the animals, our findings are otherwise consistent with prior studies with the surviving AGMs showing mild clinical disease, pathology, and prolonged viral shedding 6, 9 . The RMs in our study also exhibited mild clinical disease and pathology with shorter viral shedding from mucosal sites compared to the AGMs.

These findings show that even in the absence of severe disease both AGM and RM have utility for testing SARS-CoV-2 vaccines and therapeutics Two of the AGMs in our study developed widespread radiographic opacities and severe respiratory distress (Sp02 77%) that progressed over a 24-hour period. Taken together with the postmortem findings of diffuse alveolar damage and no evidence of congestive heart disease, these findings are consistent with a diagnosis of ARDS in both AGM1 and AGM2. Of note, both of the animals that developed ARDS did so within 24 hours following routine sampling procedures which included anesthesia and bronchoalveolar lavage. It has been our experience that these procedures are well tolerated, and procedure-related complications are exceedingly rare (fatal complication within 48-hours of procedure after 2 of 11,431 procedures in animals ranging from 1-31 years of age, unpublished data). Furthermore, AGM1 and AGM2 previously underwent the same routine sampling procedures one and three times (respectively) without J o u r n a l P r e -p r o o f complication. In our previous experience fatal complications have only occurred in animals that were severely debilitated at the time of the procedure and even in these rare cases the pathologic lesions were distinct (no evidence of diffuse alveolar disease) from the two AGMs reported herein. Therefore, in our experience, routine sampling procedures do not cause the severe COVID-19 phenotype observed in AGM1 and AGM2, even in the rare cases where fatal complications occur.

Further, dramatic increases in plasma cytokines compatible with cytokine storm were found in the aged AGMs that progressed to ARDS. Proinflammatory cytokines including TNFα, IL-1β, IL-8, IL-6, G-CSF, MCP-1, and MIP-1 have been shown to be elevated during the acute phases of acute lung injury (ALI) 26 . In human COVID-19, circulating IL-6 has been shown to correlate with radiographic abnormalities of pneumonia 3 . Indeed, overexpression of several of these cytokines were observed in both animals that progressed to ARDS. This differed from the cytokine profile in the AGMs and RMs that reached study endpoint. Interestingly, at 7 DPI all four AGMs had increased levels of IFNγ, with the two AGMs that progressed having the highest plasma concentration. Thus, elevated IFNγ in plasma could be explored as a potential predictive biomarker for advanced disease in people.

Several factors may have contributed to the severe disease phenotype observed in the AGMs in our study. Age 2 , weight 27 , and sex 2, 28 have been identified as potential predisposing factors for developing severe disease in humans. All of the AGMs included in our study were aged, with an estimated age of 16 years old. Both animals that progressed to severe disease were also female J o u r n a l P r e -p r o o f and low weight. This differs from what is reported in COVID-19 patients in which male gender 15, 29 and obesity 27 have been shown to have a higher prevalence of severe disease. The AGMs used in this infection study were also imported from nondomestic sources and although the animals were housed for 10 months at the TNPRC and deemed clinically healthy at the time of initiation of the study, there may have been historical factors that predisposed them to enhanced COVID-19 disease.

This study demonstrates that following exposure to SARS-CoV-2 aged AGMs develop a spectrum of disease, from mild to severe COVID-19, which in some cases progress to ARDS.

The cytokine expression profile in the two animals that developed ARDS is similar to that seen in the severe human disease phenotype. Our data suggest that both RM and AGM are capable of modeling mild manifestations of SARS-CoV-2 infection and that aged AGMs may additionally be capable of modeling severe disease manifestations including ARDS. 

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We would like to acknowledge Natalie Thornburg at the NCIRD for her help acquiring and characterizing the viral stock used in this infection study.