key: cord-0427973-tinubjw7
authors: Nagata, Noriyo; Iwata-Yoshikawa, Naoko; Sano, Kaori; Ainai, Akira; Shiwa, Nozomi; Shirakura, Masayuki; Kishida, Noriko; Arita, Tomoko; Suzuki, Yasushi; Harada, Toshihiko; Kawai, Yasuhiro; Ami, Yasushi; Iida, Shun; Katano, Harutaka; Fujisaki, Seiichiro; Sekizuka, Tsuyoshi; Shimizu, Hiroyuki; Suzuki, Tadaki; Hasegawa, Hideki
title: The peripheral T cell population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus 2
date: 2021-01-07
journal: bioRxiv
DOI: 10.1101/2021.01.07.425698
sha: d361588a7ee5b7d78cbf214fc94e6e1aa8d20dd4
doc_id: 427973
cord_uid: tinubjw7

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that began in December 2019. Lymphopenia is a common feature in severe cases of COVID-19; however, the role of T cell responses during infection is unclear. Here, we inoculated six cynomolgus monkeys, divided into two groups according to the CD3+ T cell population in peripheral blood, with two clinical isolates of SARS-CoV-2: one of East Asian lineage and one of European lineage. After initial infection with the isolate of East Asian lineage, all three monkeys in the CD3+ low group showed clinical symptoms, including loss of appetite, lethargy, and transient severe anemia with/without short-term fever, within 14 days post-infection (p.i.). By contrast, all three monkeys in the CD3+ high group showed mild clinical symptoms such as mild fever and loss of appetite within 4 days p.i. and then recovered. After a second inoculation with the isolate of European lineage, three of four animals in both groups showed mild clinical symptoms but recovered quickly. Hematological, immunological, and serological tests suggested that the CD3+ high and low groups mounted different immune responses during the initial and second infection stages. In both groups, anti-viral and innate immune responses were activated during the early phase of infection and re-infection. However, in the CD3+ low group, inflammatory responses, such as increased production of monocytes and neutrophils, were stronger than those in the CD3+ high group, leading to more severe immunopathology and failure to eliminate the virus. Taken together, the data suggest that the peripheral T lymphocyte population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with SARS-CoV-2. Author summary SARS-CoV-2 infection causes an illness with clinical manifestations that vary from asymptomatic or mild to severe; examples include severe pneumonia and acute respiratory distress syndrome. Lymphopenia, which is common in severe COVID-19 cases, is characterized by markedly reduced numbers of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells. Here, we showed that cynomolgus monkeys selected according to the T cell populations in peripheral blood have different outcomes after experimental infection with SARS-CoV-2. These findings will increase our understanding of disease pathogenesis and may facilitate the development of animal models for vaccine evaluation.

-16 -283 Table 2 The results revealed that the viral genome obtained from the tonsil of monkey #5404 287 after initial inoculation did not harbor any mutations (threshold = 50%). However, the viral 288 genome isolated from the lung of monkey #5412 harbored nine single nucleotide 289 polymorphisms (SNPs), including seven silent point mutations, one deletion resulting in a 290 frameshift mutation in the ORF1ab region, and a nonsynonymous mutation in ORF1b. The most 291 common base change was C > T. In addition, the genome isolated from mesenteric lymph nodes 292 from monkey #5412 harbored three SNPs, including two nonsynonymous mutations in the 293 ORF1a region and a synonymous mutation in the S region. The major sequence in these two 294 isolates was derived from WK-521, suggesting that the original inoculum replicated and resided 295 in the respiratory tract and intestine of monkey #5412, even after the second inoculation with 296 the heterologous strain. The viral genome obtained from the tonsil of monkey #5403 after the 297 second inoculation harbored a D614G mutation in the S region, suggesting the presence of 298 QH-329-037 in the tonsil after the 2nd inoculum (S3 Table) . The viral genome also obtained 299 from the tonsil of #5405 after the second inoculation harbored a D614G mutation in the S 300 region, suggesting the presence of QH-329-037 in the tonsil. Interestingly, the genome obtained 301 from the subcarinal lymph node of monkey #5405 did not harbor the D614G mutation, 302 suggesting that the original inoculum was maintained in the accessory lymph node of the lungs.

303 These results suggest that the initially inoculated virus (WK-521) was maintained in the lungs 304 and/or accessory lymph nodes, and that the second inoculated virus (QH-329-037) was 305 eliminated from the lungs of these monkeys soon after the second inoculation. 

We also determined the mutation patterns in SARS-CoV-2 isolates from the lung of 453 monkey #5412 at 6 weeks after the initial inoculation. The most common base changes were C > 

The six monkeys were anaesthetized by intramuscular injection of a mixture of 50 544 mg/mL ketamine and 20 mg/mL xylazine 

from #5404) or intestine (rectal swab from #5412) after the initial inoculation

Real-time RT-PCR confirmed viral replication in the upper respiratory tract and intestine by 193 detecting viral subgenomic mRNAs in swab samples that were positive for viral RNA

Actively-infected cells were detected in nasal swabs from two monkeys (#5403 and #5404) and 195 in a rectal swab from one monkey (#5412) (Fig. 3 and S6 Fig)

Neutralizing antibodies were detected from 10 days (monkey 202 #5399 in the CD3+ high group) or 14 days (the other three monkeys in both groups) after the 203 initial inoculation, peaking at 21 days p.i. in the CD3+ high group and 28 days p.i. in the CD3+ 204 low group (Fig. 4A)

Sidak's multiple comparisons test after application of a mixed-effects models for repeated 211 measures analysis revealed a significant difference in neutralizing antibody titers between the 212 two groups. Serum obtained from the monkeys showed cross-reactivity with both strains of -12 -213 virus

We also used in-house IgM, IgA, and IgG enzyme-linked immunosorbent assay (ELISAs) to 215 examine antibody isotypes and their binding to the spike (S), receptor binding domain (RBD), 216 and nucleocapsid (N) proteins (Fig. 4B)

IgG antibody titers increased in both groups. Spearman's correlation analysis 218 revealed that the IgA and IgG responses correlated with the neutralizing antibody response (R > 219 0.8). High levels of IgG antibodies specific for the S and RBD proteins were observed in 220 monkey #5412

Gene expression was compared between samples collected from 229 animals before (Day 0) and after (Days 1, 4, 7, R0, R1, R4, and R7) virus inoculation to identify 230 differentially expressed genes (S7A Fig). The results revealed that 331 genes were upregulated 231 significantly, while 176 genes were downregulated significantly, after virus infection. Among 232 the 507 differentially expressed genes, 78 were related to the immune response (S7B Fig). Next, 233 we conducted gene set enrichment analyses using samples collected from the CD3+ high and 234

The results revealed significant 242 upregulation of genes encoding innate anti-viral immune system-related modules (yellow dots 243 in Fig. 5B) on Days 1 and 4 in both the CD3+ high and CD3+ low groups. Of note, upregulation 244 of genes encoding inflammation-related modules (green & red dots in Fig. 5B) and 245 downregulation of genes encoding T cell-related modules (black dots in Fig. 5B) were more 246 prominent in the CD3+ low group than in the CD3+ high group

At the experimental end-point, tissue samples were also collected to detect viral

Two monkeys (#5404 and 256 #5417) euthanized at 7 days after initial inoculation had viral RNA and/or subgenomic mRNA -14

At 7 and 14 days after the second inoculation (R7d and 258 R14d), two monkeys (#5412 and #5405) from the CD3+ low group had viral RNA and/or 259 subgenomic mRNA in the lower lobe of the lungs and in the trachea. Monkey #5412 excreted 260 the virus in rectal swabs (Fig. 3) and had detectable viral RNA and/or subgenomic mRNA in the 261 large intestine and mesenteric lymph nodes at 7 days after the second inoculation. High levels of 262 viral RNA were detected in the tonsil and subcarinal lymph nodes of monkeys in both the CD3+ 263 high and low groups at various time points

TMPRSS2/VeroE6 cells in the presence of culture supernatant from the first inoculation plus 267 10% tissue homogenate

In the end, only three samples were suitable for genome 276 sequencing; the sequences obtained from these samples were compared with the Wuhan-Hu-1 the DNA Data Bank of Japan (DDBJ) Sequence Read Archive, under -15 -279 submission ID DRA011219 (BioSample accessions: SAMD00261559 -00261561). In addition, 280 because the number of reads was sufficient at the D614G position (>200), a genetic population 281 analysis of the D614G variant was performed in six samples (S3 Table). mRNA; in addition, none of the monkeys developed obvious conjunctivitis during 355 the observation period in this study. Taken together, these data suggest that a combination of the 356 intranasal and intratracheal routes (at least) might be appropriate for vaccine studies

Peripheral blood lymphocyte subsets in humans are affected by factors such as 363 gender, age, and ethnicity, and by lifestyle factors such as stress [35

Similar to 371 SARS-CoV and MERS-CoV, older age is a risk factor for severe SARS-CoV-2. Whereas 372 absolute lymphopenia is not specific to COVID-19, low CD3+, CD4+, and CD8+ T cell counts 373 in peripheral blood have been observed in severe cases of COVID-19 [39]. These cases also -21 -374 present with comorbidities such as chronic underlying diseases. Zheng et al. reported that the 375 total CD3+ count is lower in both mild and severe cases of COVID-19 than in healthy controls, 376 but that CD3+, CD8+, and NK cell counts are significantly lower in severe cases [40

Murine models of SARS-CoV and MERS-CoV infection suggest that failure to 380 induce an early IFN-I response leads to severe pathology and disease

In both groups, IRF2, 385 which regulates type I IFN production, was activated during the early phase of infection and 386 upon re-infection. However, in the CD3+ low group, inflammation overwhelmed the T cell 387 response. This is supported by the kinetics of T cell-associated cytokine and chemokine 388 production in monkey sera

T cell response, was critical for the development of more severe SARS-CoV-2 in the CD3+ low 390 group. By contrast, an early type I IFN-related innate immune response controlled viral 391 replication and dispersion at an early stage in the CD3+ high group

Levels of serum ferritin, an intracellular protein 397 that maintains iron levels, mirror the degree of inflammation in infectious diseases. In this 398 study, we did not measure ferritin levels in blood from infected monkeys; however, studies 399 show that hospitalized COVID-19 patients have high ferritin levels [2, 46]. The impact of 400 anemia and high ferritin levels on outcome after SARS-CoV-2 infection is unclear [45]. In this 401 study, one of two monkeys (#5417) with anemia that was sacrificed for planned autopsy showed 402 severe acute pneumonia and hemophagocytes in the cervical lymph nodes

Pathological evaluation revealed varying degrees of virus infection and host response 408 in the lungs of SARS-CoV-2-infected monkeys at 7 days p.i. Morphologically

Similar to SARS-CoV infection, expression of ACE2 and SARS-CoV 411 antigen-positive cells did not overlap [50]. In a severe case (monkey #5417), pulmonary edema 412 was observed, suggesting severe damage to pneumocytes. The pathological features were early 413 stage diffuse alveolar damage, with hyaline membranes and a few multinucleated giant cells, 414 similar to human cases of SARS and COVID-19

many regenerated type II cells were seen in the lungs of monkey #5417, and high levels of 419 seroconversion occurred in monkey #5412

Most infected people are asymptomatic or show mild symptoms during SARS-CoV-2 infection

421 thus some researchers wonder whether SARS-CoV-2 infection triggers protective immunity 422 against re-infection

The latest study reporting human cases of 424 COVID-19 indicate that the neutralizing antibodies against SARS-CoV-2 last only for a few 425 months [56]. The results of the present study suggest the magnitude of neutralizing antibody 426 titers in infected monkeys is dependent on disease severity, similar to human cases [56]. In 427 addition, these monkeys developed a rapid immune response against a second infection with 428 another challenge strain. NK cell and IL-17 responses, suggesting involvement of Th17 cells, 429 were stronger after the second infection than after the initial infection. Transcriptome analysis 430 revealed that upregulation of innate immune responses, rather than T and B cell responses, in 431 the CD3+ low group contributed to a marked reduction in viral replication and less severe 432 pathology, even after a second infection. Seroconversion in monkeys is common after acute 433 virus infections; indeed, we found virus-specific IgM, IgG, and IgA antibodies in the sera

IgM and IgG during SARS-CoV infection

SARS-CoV-2-specific IgG antibodies are 437 predominantly specific for the S-/RBD-and N proteins. IgG levels in symptomatic groups are by real-time RT-PCR

Subgenomic viral RNA transcripts were also detected in N 573 gene transcripts. The primer and probe sets are shown in S4 Table. Real-time RT-PCR was 574 performed using the QuantiTect Probe RT-PCR Kit (QuantiTect, Qiagen, Venlo, Netherlands) 575 and a LightCycler 480

95°C for 15 min, and 40 cycles of 94°C for 15 s and 60°C for 1 min (N1 set and the 579 sgRNA transcript primer and probe sets)

Some samples containing high viral RNA copy numbers were sent for viral sequence 581 analysis by gene analysis services

The viral genome region was amplified specifically 585 by multiplex PCR [63], and the entire sequence of the viral genome was obtained using the next 586 generation sequencer MiSeq (Illumina

Genome sequences were deposited in the DNA Data 591 Bank of Japan (DDBJ

Complete blood cell counts, hematocrit, and hemoglobin levels in peripheral blood 595 collected in EDTA tubes were measured by an autoanalyzer

Neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were measured 597 by microscopic analysis. Blood biochemistry (Glob, ALB, glucose, alkaline phosphatase (ALP), 598 and blood urea nitrogen (BUN)) of lithium-heparin treated whole blood samples was analyzed 599 using the VetScan

Cell staining was performed 604 using the NHP T/B/NK Cell Cocktail (Becton Dickinson (BD) Company, Franklin Lakes, NJ) 605 and the NHP T Lymphocyte Cocktail (BD), according to the manufacturer's instructions. After 606 treatment with BD FACS lysing solution (BD), samples were analyzed by flow cytometry using 607 a BD FACSCanto II analyzer (BD). Flow cytometry data were analyzed using FlowJo software 608

Histopathology and immunohistochemistry 611 Animals were euthanized by exsanguination under excess ketamine anesthesia and 612 then necropsied. Tissue samples were immersed in 10% phosphate-buffered formalin, 613 embedded in paraffin, sectioned, and stained with hematoxylin and eosin

Immunohistochemical analysis was performed using a polymer-based detection system 615 (Nichirei-Histofine Simple Stain Human MAX PO®

Hematoxylin was used for 621 counterstaining. The polyclonal antibody against GST-tagged N protein of SARS-CoV-2 was 622 prepared as follows: first, the recombinant N protein was constructed by inserting the N gene of 623 SARS-CoV-2 into the pGEX-6P vector

Expression of the GST-N protein of 626 SARS-CoV-2 was induced by isopropyl-D-1-thiogalactopyranoside (0.3 mM IPTG

The cell pellets were sonicated, and the inclusion bodies containing the fusion protein 628 were collected. The fusion proteins were extracted from SDS-PAGE gels after reverse staining 629 (AE-1310 EzStain Reverse

Two New Zealand White rabbits (1.5 632 kg < body weight; female

Japan) were immunized (four times at 2-week 633 intervals) with the purified protein conjugated to TiterMax Gold (Sigma-Aldrich). Rabbits were 634 sacrificed under excess anesthesia with pentobarbital sodium (64.8 mg/kg), and whole blood 635 was collected by cardiac puncture using an 18 G needle

IgG was purified from the rabbit serum using a Melon Gel IgG Spin Purification Kit

Fisher Scientific) and then used for immunohistochemistry

During the observation period, blood was obtained under anesthesia with ketamine

Serum samples were collected by centrifugation and inactivated by heating to 56C for 30 min

Serum samples were titrated (in duplicate) from 1:10 to 1:1280 in 96-well plates and reacted 643 with 100 TCID 50 of SARS-CoV

PBS-T). The well contents were 655 discarded and diluted serum samples were added to the plate. After incubation for 1 h at 37℃, 656 the plate was washed three times with PBS-T. The wells were then incubated with an 657 HRP

/5000, 50 µL/well), an HRP-conjugated goat anti-monkey IgA antibody 659 ((KPL #5220-0332, SeraCare Life Sciences, 1/5000, 50 µL/well), or an HRP-conjugated goat

anti-monkey IgG heavy and light chain antibody (A140-102P, 1/10000, 50 µL/well

) was added to the wells, and the plates were 663 incubated for 30 min at room temperature. The optical density (OD) of each well was measured 664 at 405 nm using a microplate reader

All serum samples tested in the BSL2 laboratory (all of which were confirmed 670 negative for viral RNA by RT-PCR) were irradiated for 1 min with UV-C light. Cytokine and 671 chemokine levels in monkey sera were measured using a MILLIPLEX MAP Non-Human

Primate Cytokine Magnetic Bead Panel -Premixed 23 Plex -Immunology

IL-13, IL-15, IL-17, 676 IL-18, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein 1 alpha 677 (MIP-1α), MIP-1β, sCD40L, transforming growth factor alpha (TGF-α)

Whole blood was collected from animals at multiple time points using PAXgene

RNA was extracted using PAXgene Blood RNA Kits (PreAnalytiX) and 686 shipped to Macrogen Corp. Japan (Kyoto, Japan) for NGS sequencing. Next, cDNA libraries 687 were prepared using a TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina) in 688 accordance with the TruSeq Stranded Total RNA Sample Prep Guide (Part #15031048 Rev. E 689 protocol). Next, the cDNA libraries were paired-end sequenced (read length = 101 bp) on a 690 NovaSeq6000 sequencer (Illumina). Raw FASTQ files were quality checked using fastqc 691 v0

Ensembl release 693 101) using the STAR aligner v2.7.3a [69] and default settings. Read fragments (paired reads 694 only) were quantified per gene per sample using featureCounts v1

Significantly differentially expressed genes between samples collected 698 before and after virus infection were identified using DESeq2 v1.26.0 [71] with default settings, 699 and a minimum adjusted P-value significance threshold of 0.05. Volcano plots were created 700 from shrunken log2-fold change values for each gene, calculated by DESeq2 (shrinkage type: 701 normal). For the heatmaps, DESeq2-normalized counts per gene were plotted using the heatmap between samples collected from animals in the CD3+ high and low groups after virus 706 infection

Data are expressed as the mean and standard error of the mean. Statistical analyses 710 were performed using Graph Pad Prism 8 software

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We thank Dr Shutoku Matsuyama and Dr Makoto Takeda

Infectious Disease) for providing VeroE6-TMPRSS2 cells and SARS-CoV-2 isolates. We also 720 thank Dr Masayuki Shimojima, Dr. Hideki Asanuma, Dr Makoto Kuroda

Japan) for helpful discussion. We also thank our colleagues at the Institute, especially

Ms Takiko Yoshida, Dr Michiyo Kataoka, Dr. Dai Izawa, and Ms Yuriko 725 Suzaki, for technical assistance

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Trimmomatic: a flexible trimmer for

Illumina sequence data

STAR: 997 ultrafast universal RNA-seq aligner

featureCounts: an efficient general purpose 1001 program for assigning sequence reads to genomic features

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was funded through the Research Program on Emerging and Reemerging Infectious 1022 Diseases from the Japan Agency for Medical Research and Development (JP19fk0108072) and 1023 by a grant-in-aid for scientific research from the Ministry of Education

Science, and Technology in Japan (18H02665). H.S. was funded through the Japan Agency for 1025 Medical Research and Development (JP19fk0108084). T. Su was funded through the Japan 1026 Agency for Medical Research and Development (JP19fk0108104, JP20fk0108104, and 1027 JP19fk0108110). H.H. was funded through the Japan Agency for Medical

The funders played no role in study design, data collection and 1029 analysis, decision to publish, or preparation of the manuscript

HH 1044 Supervision: NN, T. Su, HH 1045 Validation: NN, T. Su, HH 1046 Visualization: NN, KS 1047 Writing -Original Draft

After assigning animals to "CD3+ high" and "CD3+ low" groups, six 1057 cynomolgus monkeys were infected with an isolate from East Asia (WK-521 strain) via a 1058 combination of intranasal (0.125 mL, sprayed into the right nostril), conjunctival (0.1 mL, 1059 dropped into the right eye), and intratracheal (1.275 mL virus solution plus 2 mL saline via a 1060 catheter) inoculation. After the initial inoculation, body weight was measured, and samples were 1061 collected at various time points (blue arrows). Red arrows denote autopsy at 7 or 14 days after 1062 the first or second inoculation (n = 1 per group at each time point). Four monkeys received a 1063 second inoculation with an isolate from Europe (QH-329-037 strain). (B) Clinical scores of 1064 cynomolgus monkeys inoculated with SARS-CoV-2. Cool (blue and aqua) and warm (red and 1065 orange) colored symbols and lines indicate data from the CD3+ high group and CD3+ low 1066 group animals, respectively. After transfer to the ABSL3 facility, the monkeys were observed 1067 once daily for clinical signs and scored accordingly

Representative cytokines 1081 were profiled by multiplex analysis. Assays were performed using unicate samples at each time 1082 point. Cool (blue and aqua) and warm (red and orange) colored symbols and lines indicate data 1083 from the CD3+ high and CD3+ low groups, respectively. Each dot/line represents data from an 1084 individual animal

Cool 1089 (blue and aqua) and warm (red and orange) colored bars indicate data from CD3+ high and 1090 CD3+ low groups, respectively. Each bar represents data from an individual animal. After initial 1091 viral inoculation with the WK-521 strain, clinical samples (conjunctiva, nasal, throat, and rectal 1092 swabs) were collected. The second inoculation with QH-329-037 strain was performed 35 days 1093 after the first inoculation. + indicates samples that were positive for subgenomic mRNA (black) 1094 or virus (red)

Antibody subclasses and specificity for the spike (S), 1100 receptor binding domain (RBD), and nucleocapsid (N) proteins were assessed using in-house

Cool (blue and aqua) and warm (red and orange) colored 1102 symbols and lines indicate data from the CD3+ high and CD3+ low groups, respectively. Each 1103 dot/line represents data from an individual animal. R, correlation coefficient (Spearman's 1104 correlation analysis) between the neutralization and ELISA tests

The name of each significantly enriched module is listed, along with the module ID (in 1111 brackets) (P < 0.01). Green and gray dots indicate inflammation-and B cell response-related 1112 modules, respectively. Red and blue indicate the proportion of genes in a particular module that 1113 is upregulated or downregulated in the CD3+ high group compared with the CD3+ low group. 1114 Each module is represented by a box, where the width is proportional to the effect size 1115 (AUROC value calculated from the number of genes in the module and ranking by the Cerno 1116 test), while brighter colors indicate lower P-values. Gene set enrichment analysis in the CD3+ -51 -1117 high group (left panel) and CD3+ low group

Day 0) (B). The name of each significantly enriched module name is listed along with module

Yellow, green & red, gray, and black dots indicate modules related 1121 to innate immunity, inflammation, CD4+ T cell response, and T & NK cell responses, 1122 respectively. Red and blue indicate the proportion of genes in a particular module that is 1123 upregulated or

Each module is represented as a pie chart, where the size is proportional to the effect size 1125 (AUROC value calculated from the number of genes in the module and ranking by the Cerno 1126 test), while brighter colors indicate lower P-values

Detection of virus RNA in tissue samples from cynomolgus monkeys 1129 inoculated with SARS-CoV-2. Tissue samples were obtained from monkeys at 7 days 1130 post-inoculation with WK-521 strain (#5404 and #5417), and at 7 days (#5403 and #5412) or 14 1131 days (#5399 and #5405) after re-infection with QH-329-037 strain. Cool (blue and aqua) and 1132 warm (red and orange) colored bars indicate data from the CD3+ high group and CD3+ low 1133 group, respectively

Pathology of cynomolgus monkeys inoculated with SARS-CoV-2. (A) Gross 1136 pathology of lungs from monkeys at 7 days post-inoculation with WK-521 strain (#5404 and 1137 #5417), and at 7 days (#5403 and #5412) or 14 days (#5399 and #5405) after re-infection with

Collections of mononuclear cells were seen in the 1144 airspaces of the middle lobe of the right lung of monkey #5404 (B, upper row). Pulmonary 1145 edema with polymorphonuclear leukocyte infiltration and proliferating type II cells overlying 1146 pulmonary walls were observed in the lower lobe of the right lung of monkey #5417 (B, lower 1147 row)

Hematoxylin and eosin staining (H&E). (C) Double immunohistochemistry identified cell 1149 collections in alveolar air spaces at 7 days after the initial inoculation (upper row from #5404

An anti-CD68 rabbit polyclonal antibody (brown) and an 1152 anti-CD3-monoclonal antibody (green) were used for IHC in C. 1153 <3.4 kg. Each dot represents data from an individual animal. The blue and red colored 1158 symbols denote data from the CD3+ high and low groups

Biochemical 1162 markers including globulin (Glob), albumin (ALB), glucose, alkaline phosphatase (ALP), and 1163 blood urea nitrogen (BUN) in lithium-heparin treated whole blood samples were measured at 1164 various time points after inoculation (B)

The brown dashed line on the vertical axis indicates the day of second inoculation

Variations in deep body temperature detected by the temperature logger

Black arrows, 1171 animal transfer date (under anesthesia) from the animal facility to the animal biosafety level 3 1172 (ABSL3); red and yellow arrow heads, virus inoculation under anesthesia with a mixture of 1173 ketamine and xylazine; Red brace, deviation from diurnal variation indicates high fever. The 1174 fluctuation of deep body temperature within a day was maintained during ABSL3 1175 acclimatization

Absolute white blood cell (WBC) count, including total WBC, 1182 neutrophils, eosinophils, and basophils, in EDTA-treated whole blood samples was measured at -54 -1183 various time points after inoculation (B). Markers of leukocyte differentiation, CD4 and CD8, 1184 were detected by flow cytometry at various time points after inoculation (C). Cool (blue and 1185 aqua) and warm (red and orange) colored symbols and lines indicate data from the CD3+ high 1186 and CD3+ low groups

Cytokine and chemokine levels in serum samples from cynomolgus 1190 monkeys inoculated with SARS-CoV-2. Sera were obtained from six monkeys at various 1191 time points after inoculation. Pro-inflammatory cytokines and chemokines (A), helper T 1192 cell-related cytokines (B), and other representative factors in serum that drive proliferation of 1193 epithelial cells (TGF-α) and neutrophils (IL-8) (C) were profiled by multiplex analysis. Assays 1194 were performed using unicate samples per time point. Cool (blue and aqua) and warm (red and 1195 orange) colored symbols and lines indicate data from the CD3+ high and CD3+ low groups, 1196 respectively. Each dot/line represents data from an individual animal

Detection of subgenomic RNA in clinical samples and tissue samples from 1200 cynomolgus monkeys inoculated with SARS-CoV-2. Virus RNA-positive samples from

Figures 3 and 6 were re-examined to detect viral RNA and subgenomic RNA using three primer 1202 sets (A and B, respectively)

Transcriptome analysis of blood samples from cynomolgus monkeys -55 -1205 inoculated with SARS-CoV-2. Volcano plot showing the magnitude and significance of 1206 differentially expressed genes between samples collected from animals before (Day 0) and after 1207 (Days 1, 4, 7, R0, R1, R4, and R7) virus infection (A)

Plots shown in brighter red or blue represent genes that were either upregulated (190/331 genes) 1211 or downregulated (86/176 genes) by more than 2-fold. Expression of immunity-related genes in 1212 peripheral whole blood samples collected from animals before

Heatmaps showing normalized counts per gene, scaled 1214 by rows of 78 immune-related genes among the 507 genes significantly upregulated or 1215 downregulated by virus infection (adjusted P-value < 0.05)

Double immunohistochemistry to detect virus antigens (brown) and ACE2

1221 (green) in the lungs at 7 days after initial inoculation. ACE2 was detected in the intact 1222 brush border of the respiratory epithelia in the intrapulmonary bronchus

Viral antigen was 1224 detected in linear pneumocytes and type I pneumocytes (red arrow), and slight expression of 1225 ACE2 was detected in round pneumocytes (suggestive of type II pneumocytes) (black arrows), 1226 in the alveolar area in the absence of inflammatory infiltration (upper row, right)

Lung pathology in cynomolgus monkeys receiving a second inoculation 1234 with SARS-CoV-2. Representative histopathology images of lungs from monkeys obtained at 1235 7 days (#5403 and #5412) or 14 days (#5399 and #5405) after re-infection with

Cellular infiltration, including lymphocytes and macrophages, can be seen around the 1237 bronchi and in the alveoli in the middle lobe of the right lung from monkey #5403

Lymphoid aggregates around 1240 small vessels (red arrowheads) and fibrotic inflammation with lymphocyte aggregation in the 1241 alveolar area and pleura (blue arrowheads) were seen in the right lung from monkeys #5412 and 1242 #5405 (third and fourth rows). Scale bars: 500 µm

Representative images of histopathological lesions from cynomolgus 1246 monkeys after experimental inoculation with SARS-CoV-2. Representative 1247 hemophagocytosis images of the lung and lymph nodes from monkey #5417 obtained at 7 days 1248 after infection with WK-521 strain (A). Hemophagocytes are seen in the alveoli and sinus of

Hematoxylin and eosin (H&E) staining. Eosinophil (yellow arrows) and plasma 1251 cell (blue arrows) infiltration into the mesenteric lymph node and intestines from monkey #5412 1252 at 7 days after the second inoculation with QH-329-037 (B). Cellular infiltration, including 1253 eosinophils and plasma cells, can be seen in the sinus of the mesenteric lymph node and the 1254 lamina propria of the small and large intestine

Cross neutralization of two strains of SARS-CoV-2 in monkey sera after 1259 experimental infection

Summary of the results of next generation sequencing of SARS-CoV-2