key: cord-0948451-gugfjhoi
authors: Ishigaki, Hirohito; Nakayama, Misako; Kitagawa, Yoshinori; Nguyen, Cong Thanh; Hayashi, Kaori; Shiohara, Masanori; Gotoh, Bin; Itoh, Yasushi
title: Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques
date: 2020-12-29
journal: Virology
DOI: 10.1016/j.virol.2020.12.013
sha: e0143025f8cac9fa227c5905973c90f764e6d7f1
doc_id: 948451
cord_uid: gugfjhoi

We examined the pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in cynomolgus macaques for 28 days to establish an animal model of COVID-19 for the development of vaccines and antiviral drugs. Cynomolgus macaques infected with SARS-CoV-2 showed body temperature rises and X-ray radiographic pneumonia without life-threatening clinical signs of disease. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes producing interferon (IFN)-γ specifically for SARS-CoV-2 N-protein were detected on day 14 in one of three macaques with viral pneumonia. In the other two macaques, in which a neutralizing antibody was not detected, T-lymphocytes producing IFN-γ specifically for SARS-CoV-2 N protein increased on day 7 to day 14, suggesting that not only a neutralizing antibody but also cellular immunity has a role in the elimination of SARS-CoV-2. Thus, because of similar symptoms to approximately 80% of patients, cynomolgus macaques are appropriate to extrapolate the efficacy of vaccines and antiviral drugs for humans.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been spreading around the world since late 2019 (Zhu et al., 2020) , and WHO declared a pandemic on March 11, 2020. Accumulating reports indicate varying degrees of illness including asymptomatic patients, patients with mild respiratory symptoms, and patients with acute respiratory distress syndrome (ARDS) requiring admission to an intensive care unit (ICU) Guan et al., 2020; . In addition to the development of vaccines and antiviral drugs specific for SARS-CoV-2, determination of the pathogenicity in patients with severe clinical signs of disease and development of therapeutics for severe cases are urgent issues.

For the development of prophylactics and therapeutics for SARS-CoV-2 infection, not only in vitro studies but also in vivo studies are required for evaluation of their efficacy, especially estimation of in vivo efficacy, for which assessments with challenge infection are difficult in clinical trials. Therefore, animal models that show pathogenicity similar to that in humans are necessary for research and development of vaccines and antiviral drugs (Cleary et al., 2020) . The results of several studies on experimental infection of SARS-CoV-2 in animals have been reported. In a mouse model, SARS-CoV-2 propagated in the lungs of human angiotensin-converting enzyme 2 (ACE2) transgenic mice but not in the lungs of wild-type mice, and the virus caused interstitial pneumonia in the ACE2 transgenic mice . However, co-expression of human ACE2 and endogenous mouse ACE2 may change the disease progression to recapitulate COVID-19.

Wild-type Syrian hamsters are sensitive to SARS-CoV-2, which propagated in the lungs to cause viral pneumonia, indicating a useful small animal model (Chan et al., 2020; Imai et al., 2020) .

However, since the viral pneumonia was resolved within 2 weeks in Syrian hamsters and antibodies J o u r n a l P r e -p r o o f that react to hamster molecules in order to examine immune responses are not available, another model is required to examine the pathogenicity of severe COVID-19. SARS-CoV-2 also propagated and caused lung inflammation and pneumonia in rhesus and cynomolgus macaques (Yu et al., 2020; Williamson et al., 2020; Rockx et al., 2020; Munster et al., 2020; Deng et al., 2020) . The pathogenicity in macaques was examined until 21 days after virus infection in all of the studies except for one study , in which the reason for the prolonged detection of viral genes in patients and virus antigen specific-T-lymphocyte responses were not revealed. Therefore, in the present study, we observed cynomolgus macaques infected with SARS-CoV-2 for 4 weeks and examined T-lymphocyte responses specific for SARS-CoV-2 antigen peptides.

The macaque model, of which immune responses and metabolism resemble those of humans, is useful to extrapolate the efficacy of vaccines and antiviral drugs in humans against SARS-CoV-2. In our previous studies on influenza virus infection, various influenza viruses including pandemic and avian influenza viruses propagated in cynomolgus macaques that showed clinical signs of disease similar to human symptoms (Itoh et al., 2009; Muramoto et al., 2014) . In addition, we detected influenza viruses that were less sensitive to neuraminidase inhibitors in treated macaques, indicating a useful model for predicting the emergence of a drug-resistant virus (Itoh et al., 2015; Suzuki et al., 2020) . Therefore, we have used the cynomolgus macaque model to evaluate the efficacy of vaccines and antiviral drugs in influenza virus infection (Arikata et al., 2012; Nakayama et al., 2013; Kitano et al., 2014; Arikata et al., 2019; Nguyen et al., 2020) . In the present study, we expanded our experimental system to establish a SARS-CoV-2 infection model in cynomolgus macaques for preclinical studies.

We revealed the pathogenicity of SARS-CoV-2 in the cynomolgus macaques.

SARS-CoV-2 propagated in respiratory tissues and caused body temperature rises in all of the macaques. However, viral pneumonia in X-ray radiographs was confirmed in one macaque, in which a neutralizing antibody against SARS-CoV-2 in plasma was detected. We also found a thrombus in the lung of a macaque infected with SARS-CoV-2 as reported in human cases (Wichmann et al., 2020) . These results are similar to observations in human patients with COVID-19 (Zhu et al., 2020) . Compared to influenza virus infection, the rate of detection of a neutralizing antibody was low in macaques infected with SARS-CoV-2 (Arikata et al., 2012; Wang et al., 2020b) . In addition, we examined T-lymphocyte responses specific for SARS-CoV-2 antigen, which were not analyzed in the other macaque studies. Interferon (IFN)-γ responses with delayed interleukin (IL)-2 responses in antigen-specific T-lymphocytes might be related to virus elimination without a neutralizing antibody. Thus, cynomolgus macaques are an appropriate animal model of SARS-CoV-2 infection for the development of vaccines and antiviral drugs.

We inoculated the SARS-CoV-2 JPN/TY/WK-521/2020 (WK-521) strain (Matsuyama et al., 2020) into the conjunctiva, nasal cavity, oral cavity, and trachea of three cynomolgus macaques to examine the pathogenicity of the strain. We collected swab samples to examine virus propagation in the macaques. The virus was detected in swab samples of the conjunctiva, nasal cavity, oral cavity, and trachea of all of the macaques on day 1 (the day after virus inoculation) ( Table 1 ). The J o u r n a l P r e -p r o o f virus was detected in nasal and oral swab samples of two macaques, CE0202M and CE0324F, until day 7. No infectious virus was detected in swab samples after day 10 or in homogenized tissue samples collected on day 28 at autopsy (listed in Table S1 ). On the other hand, a large number of viral RNA in the swab samples was detected on day 1 and gradually decreased until day 28 (Fig.   S1 ). The SARS-CoV-2 N gene was detected in respiratory and rectal samples of CE0202M until day 28. In CE0324F, a low copy number of the viral gene in the nasal swab was detected on day 28.

In the nasal swab sample of CE1242F, approximately 10 4 gene copies/mL were detected on day 28.

The results of viral gene detection were consistent with prolonged detection of viral genes in human specimens (Shi D et al, 2020) .

Cynomolgus macaques showed clinical signs of disease after infection with SARS-CoV-2 WK-521. All of the macaques showed significant rises in body temperature after virus inoculation (day 0 to day 3) (Fig. 1 ). Body temperatures of two macaques (CE0324F and CE1242F) on day 1 were higher than 39.5 o C (Fig. 1B , C) and average temperatures at nighttime from day 0 to day 3 were significantly higher than that on day -1 (Fig. 1E , F). On the other hand, CE0202M showed a significant rise in body temperature only at night on day 0, day 1 and day 3 (Fig. 1A, D) . Body weight of CE0324F decreased by 10% until day 28 due to loss of appetite (Fig. S2A) . No macaques showed clinical scores that reached a humane endpoint (Table S2) . Therefore, infection with WK-521 induced mild clinical signs of disease that were varied in the macaques.

J o u r n a l P r e -p r o o f Viral pneumonia and/or bronchopneumonia were observed in cynomolgus macaques infected with SARS-CoV-2. Chest X-ray radiographs revealed a ground glass appearance in the lungs, especially in the peripheral pulmonary areas, of CE0324F from day 3 to day 7 but not after day 10 ( Fig. 2A-C) . Saturation of percutaneous oxygen (SpO 2 ) values in infected macaques at each sampling were higher than 90% during the study (Fig. S2B ). At autopsy on day 28, demarcated bright redness was observed on the surface of the right lower lung of CE0202M ( 

The blood cell population in macaques infected with SARS-CoV-2 was examined. The numbers of total white blood cells and granulocytes in CE0202M and CD1242F were increased temporally on day 1, but the number of total white blood cells in CE0324F was decreased on days 1 and 3 and it was increased after day 14 ( Fig. 5A , B). The numbers of lymphocytes in the blood of CE0324F and CE1242F were decreased on day 1 and returned to the levels before infection on day 7 to day 10 ( Fig. 5C ), whereas the numbers of monocytes in CE0324F and CE1242F were increased on day 7 and day 3, respectively ( Fig. 5D ). The numbers of platelets in CE0324F and CE1242F were decreased on day 3 (Fig. 5E ). Thus, two of the three macaques (CE0324F and CE1242F) showed apparent changes in the population of blood cells after SARS-CoV-2 infection.

Immune responses against SARS-CoV-2 in the cynomolgus macaques were examined. CE0324F showed marked changes in plasma cytokine levels after infection (Fig. 6) . The levels of the inflammatory cytokine interleukin-6 (IL-6) together with levels of monocyte chemotactic protein-1 (MCP-1) and IL-10 on day 1 were higher than those before infection. Levels of the Th2 cytokines IL-4 and IL-13, Th17 cytokine IL-17, and chemokine MIP-1α were increased 10 days after virus infection and the level of IL-8 was increased 21 days after virus infection, although the changes in IL-17 and MIP-1α were small. Levels of the Th1 cytokines interferon-γ (IFN-γ) and IL-12 were increased on day 1 after infection. IL-2 and TNF-α were detected in the plasma of CE0324F before infection (day 0). CE1242F showed low cytokine responses in IL-6, MCP-1, IL-10, IL-4, IL-13, MIP-1α, IFN-γ, and IL-12 compared with those in CE0324F. In CE0202M, levels of IL-8 and IL-12 on day 3 and levels of IL-13 from day 10 to day 17 were transiently increased, but no apparent changes in other cytokines were detected during the study.

A neutralizing antibody against SARS-CoV-2 was detected in one macaque. A low titer of the neutralizing antibody against WK-521 was detected in CE0324F on day 10 after virus inoculation, but no neutralizing antibody was detected in the other two macaques until day 28 (Table 2 ). An IgG response against SARS-CoV-2 S and N proteins was detected in the plasma of CE0324F and CE1242F after day 14 and day 21, respectively, using an available antibody detection kit for human IgG/IgM, which seemed not to react to monkey IgM (Fig. S4) . No antibody response against SARS-CoV-2 was detected in CE0202M, in which the number of BALTs was smaller than those in CE0324F and CE1242F (Fig. S3C) , indicating a relationship between IgG responses and BALT formation.

J o u r n a l P r e -p r o o f T lymphocyte responses specific for SARS-CoV-2 N protein and S protein were examined. In CE0324F, in which a neutralizing antibody was detected, increases in the numbers of IL-2 and IFN-γ-producing cells specific for SARS-CoV-2 N protein were seen on day 14 after virus inoculation (Fig. 7) . In CE0202M, increases in the numbers of IFN-γ-producing cells and IL-2-producing cells were detected on day 14 and day 21, respectively. In CE1242F, increases in the numbers of IFN-γ-producing cells and IL-2-producing cells were detected on day 7 and day 14, respectively. In the two latter monkeys, the increase in the number of IL-2-producing cells followed that of IFN-γ-producing cells and the stimulation indices of IFN-γ-producing cells were higher than those of IL-2-producing cells. Stimulation indices of IFN-γ-producing cells and IL-2-producing cells specific for SARS-CoV-2 S protein peptides were increased on day 28 compared with those before infection, but stimulation indices of IFN-γ-producing cells specific for SARS-CoV-2 S protein peptides were lower than those of IFN-γ-producing cells specific for SARS-CoV-2 N protein peptides (Fig. S5 ).

We revealed the pathogenicity of SARS-CoV-2 in cynomolgus macaques to establish an animal model for the development of vaccines and antiviral drugs. SARS-CoV-2 was detected predominantly in the nasal cavity and oral cavity of cynomolgus macaques for one week.

Furthermore, infection with SARS-CoV-2 caused a body temperature rise in all of the cynomolgus macaques examined. However, radiographic viral pneumonia was observed in one macaque, in which a neutralizing antibody against SARS-CoV-2 was detected. Histologically, pneumonia in the repairing phase and/or bronchopneumonia were observed in all of the macaques and a thrombus in a J o u r n a l P r e -p r o o f pulmonary blood vessel was detected (Wichmann et al., 2020) . Mild clinical signs of disease in the cynomolgus macaques in the present study are consistent with the results of previous studies using cynomolgus macaques and rhesus macaques (Rockx et al., 2020; Munster et al., 2020) as well as the results of a clinical study showing that approximately 80% of human patients with COVID-19 did not show critical disease (Wiersinga et al., 2020) . In addition, virus propagation in the respiratory tissue of cynomolgus macaques was confirmed, indicating that a cynomolgus macaque model is a suitable model for evaluation of the efficacy of vaccines and antiviral drugs.

Plasma cytokine and chemokine responses were detected after infection with WK-521 in the cynomolgus macaques. The level of the representative inflammatory cytokine IL-6 in CE0324F, in which a neutralizing antibody was detected, was increased on day 1 after virus infection, but the increase in the level of this cytokine was transient at an early stage of infection and the inflammatory cytokine TNF-α was not increased at an early stage but was increased on day 17 in CE0324F. Therefore, no evidence of a cytokine storm or systemic inflammatory response syndrome was seen in the present study. On the other hand, a response of IL-4, a representative cytokine of a Th2 response, in plasma of CE0324F and a weak response of IL-13 in plasma of CE0202M and CD0324F were detected after day 10. Thus, the IL-4 response at a late phase of SARS-CoV-2 infection may be related to antibody production. An IL-17 response in plasma of CE0324F was detected at a late phase of SARS-CoV-2 infection, but the level was too low to discuss a role of IL-17 in antibody production.

No neutralizing antibody against SARS-CoV-2 was detected 28 days after virus inoculation in two macaques in which body temperature increased and the virus propagated. On the other hand, antibody responses specific for SARS-CoV-2 antigens were detected in most of the J o u r n a l P r e -p r o o f macaques on day 14 after infection in previous studies (Rockx et al., 2020; Munster et al., 2020) .

The difference between our study and the other studies in the antibody response rate might be related to the age of macaques since some aged macaques (15 to 20 years of age) showed a weak antibody response against SARS-CoV-2 antigen in one of the previous studies (Rockx et al., 2020) .

Our results are consistent with the results of studies showing delayed IgG responses in human patients with COVID-19 Qu et al., 2020; Shen et al., 2020) . The results of the present study indicate that delayed antibody responses would occur in patients without severe symptoms and complications. The results also suggested that the lack of an antibody against SARS-CoV-2 does not necessarily mean evidence of uninfected individuals.

We also examined T-lymphocyte responses specific for SARS-CoV-2 N and S protein peptides. In macaque CE0324F, in which a neutralizing antibody was detected, the number of cells producing IFN-γ and IL-2 specific for SARS-CoV-2 N protein was increased 2 weeks after virus inoculation. On the other hand, in CE0202M and CE1242F, in which no neutralizing antibody was detected, an increase in the number of cells producing IL-2 specific for SARS-CoV-2 N protein followed an increase in the number of cells producing IFN-γ. The results indicate that an IFN-γ response with a low IL-2 response is not sufficient for induction of a neutralizing antibody. In addition, the IFN-γ response (Th1 response) that helps so-called 'cellular immunity' might contribute to elimination of SARS-CoV-2 without a neutralizing antibody. Another possibility is that the early IFN-γ response in CE1242F was induced by memory Th1 cells, suggesting previous exposure to other coronaviruses. The effects of immunity against the common cold coronavirus on SARS-CoV-2 infection will be analyzed using the macaque model in the future (Sette and Crotty 2020).

The viral genes of SARS-CoV-2 were detected after day 10, although no infectious virus was detected in swab samples. Two possible reasons are thought to explain the discrepancy as seen in influenza B virus (Kitano et al., 2010) . One is that the limit to detect infectious viruses using the cell culture was higher than that to detect the viral RNA using RT-PCR. The other is that viral genes and/or gene fragments were released from the infected cells without production of infectious viruses. Therefore, in macaques CE1242F and CE0202M that showed a low level and no detectable level of IgG responses against SARS-CoV-2 antigen without a detectable neutralizing antibody, very low levels of infectious virus and/or infected cells in the nasal cavity and the rectum might not be eliminated by the specific antibody until day 28.

The level of amylase in the plasma of CE0324F was temporally increased. It has been reported that blood amylase was released from the injured pancreas in patients with COVID-19 pneumonia (Wang et al., 2020a; . However, we did not detect inflammation or necrosis in the pancreas of macaques infected with SARS-CoV-2 at autopsy in the present study (data not shown). On the other hand, lymphoid infiltration in the submandibular gland of CE0324F might be related to the increase of plasma amylase (Liu et al., 2011) , indicating that plasma amylase was derived from infected salivary glands and that the virus detection in oral swab samples was due to virus propagation in the salivary glands , although a viral antigen was not detected in immunohistochemical staining of the salivary glands on day 28, probably due to disappearance of the virus antigen before autopsy (data not shown).

In comparison with other macaque studies, rhesus macaques showed visible clinical signs of disease after SARS-CoV-2 infection such as piloerection, hunched posture, and pale appearance (Munster et al., 2020) , which were not noticed in cynomolgus macaques in the present study and the previous study (Rockx et al., 2020) . In the present study, significant body temperature rises in all of the cynomolgus macaques, especially at night, were recorded using a telemetry recording system, and such significant body temperature rises were not observed in the other studies including studies using rhesus macaques. Histological pneumonia was also detected in all of the infected cynomolgus macaques in the present study. In addition, virus propagation in rhesus macaques, especially the duration of virus detection, was similar to that in cynomolgus macaques. Therefore, the cynomolgus macaque model is also thought to be a suitable animal model for evaluating the efficacy of vaccines and antiviral drug candidates for SARS-CoV-2 infection.

In the present study, we revealed that SARS-CoV-2 propagated in the respiratory tissues of cynomolgus macaques and that SARS-CoV-2 caused clinical signs of disease, indicating that the cynomolgus macaque model is useful for the evaluation of vaccination and therapies. In addition, delayed and low antibody responses in the macaque model may explain the prolonged virus shedding and reinfection in human patients .

This study was carried out in strict accordance with the Guidelines for the Husbandry and Committee (permit number: 2020-4-2). Regular veterinary care and monitoring, balanced nutrition and environmental enrichment were provided by the Research Center for Animal Life Science at Shiga University of Medical Science. The macaques were euthanized at the endpoint (28 days after virus inoculation) using ketamine/xylazine followed by intravenous injection of pentobarbital (200 mg/kg). The animals were monitored every day during the study to be clinically scored as shown in Table S2 and to undergo veterinary examinations to help alleviate suffering. Animals would be euthanized if their clinical score reached 15 (a humane endpoint).

Fifteen-year-old and ten-year-old female cynomolgus macaques (CE0324F and CE1242F) and a fifteen-year-old male cynomolgus macaque (CE0202M) were born at Shiga University of Medical Science from maternal parent macaques that originated from Indonesia and paternal parent macaques that originated from Vietnam. All procedures were performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering. Food pellets of CMK-2 (CLEA Japan, Inc., Tokyo, Japan) were provided once a day after recovery from anesthesia and drinking water was available ad libitum. The animals were singly housed in cages equipped with bars to climb up for environmental enrichment under controlled conditions of humidity (39% -61%), temperature (24 -26°C), and light (12-h light/12-h dark cycle, lights on at 8:00 a.m.). Two weeks before virus inoculation, a telemetry probe (M00, Data Sciences International, St. Paul, MN) was implanted in the peritoneal cavity or subcutaneous tissue of each macaque under ketamine/xylazine anesthesia followed by isoflurane inhalation to monitor body temperature. The

The macaques were challenged with the WK-521 virus (2.2 × 10 6 TCID 50 ) inoculated into the conjunctiva (0.05 mL × 2), nostrils (0.5 mL × 2), oral cavity (0.9 mL), and trachea (5 mL 

Virus neutralization assay Plasma samples were pretreated with a receptor-destroying enzyme (RDEII, Denka Seiken, Tokyo, Japan) at 37°C overnight and then inactivated at 56°C for 1 h. The diluted samples were mixed with 100 TCID 50 /well of the WK-521 strain for 30 min. Then the mixture was added onto a VeroE6 monolayer in 96-well plates. After 1-h incubation, the cells were cultured in MEM containing 0.1% BSA and 5 µg/mL trypsin. After incubation at 37°C for 6 days, the number of wells with cytopathic effects was counted in quadruplicate culture. Neutralization titers were expressed as the dilution in which cytopathic effects were observed in 50% of the wells.

Plasma was collected on indicated days. A COVID-19 IgG/IgM immunodetection kit (Novus Biologicals USA, Centennial, CO) was used for detection of IgG and IgM specific for SARS-CoV-2 S and N proteins. Monkey IgM seemed not to react to the human IgM detection system.

Peripheral blood mononuclear cells after separation from red blood cells were stored at -80°C until use. Thawed cells (5 × 10 5 /well) were cultured with a peptide pool of SARS-CoV-2 N protein and S protein (0.6 nmol/mL) (PepTivator, Miltenyi Biotech, Bergisch Gladbach, Germany) in the presence of anti-CD28 antibody (0.1 µg/mL) overnight in ELISPOT plates coated with anti-IFN-γ and IL-2 antibodies (Cellular Technology Limited, Shaker Heights, OH). The number of cytokine-producing cells was counted according to the manufacturer's instructions. after virus inoculation. Average body temperatures on each day were compared with those of day -1 (from 8 p.m. on day -1 to 8 a.m. on day 0 before virus inoculation). Asterisks indicate significant differences from average temperature on day -1 (p < 0.05, Student's t-test).

(A-C) X-ray radiographs of CE0324F taken on day 0 before infection (A) and day 3 (B) and day 7 (C) after virus inoculation. Red circles indicate ground glass appearance (pneumonia). (D-F) Lung macroscopic appearance. All of the macaques infected with the virus were autopsied 28 days after virus inoculation. White circles indicate lesions with a bright red or dark red/brown color. 

Biochemical analysis was performed using plasma collected on the indicated days after virus inoculation: (A) total bilirubin, (B) alanine aminotransferase, (C) alkaline phosphatase, (D) blood urea nitrogen, (E) creatinine, and (F) amylase.

Peripheral blood cells were collected on the indicated days after virus inoculation. The concentrations of (A) total white blood cells, (B) granulocytes, (C) lymphocytes, (D) monocytes, and (E) platelets were determined. 

Memory immune responses against pandemic (H1N1) 2009 influenza virus induced by a whole particle vaccine in cynomolgus monkeys carrying Mafa-A1*052:02

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Establishment of a cynomolgus macaque model of influenza B virus infection

Efficacy of repeated intravenous administration of peramivir against highly pathogenic avian influenza A (H5N1) virus in cynomolgus macaques

Epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Respiratory disease in rhesus macaques inoculated with SARS-CoV-2

Disease severity is associated with differential gene expression at the early and late phases of infection in nonhuman primates infected with different H5N1 highly pathogenic avian influenza viruses

Protection against H5N1 highly pathogenic avian and pandemic (H1N1) 2009 influenza virus infection in cynomolgus monkeys by an inactivated H5N1 whole particle vaccine

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A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

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Delayed specific IgM antibody responses observed among COVID-19 patients with severe progression

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Clinical Characteristics and Outcomes of Coronavirus Disease 2019 Among Patients With Preexisting Liver Disease in the United States: A Multicenter Research Network Study

Low replicative fitness of neuraminidase inhibitor-resistant H7N9 avian influenza a virus with R292K substitution in neuraminidase in cynomolgus macaques compared with I222T substitution

Pancreatic injury patterns in patients with coronavirus disease 19 pneumonia

Kinetics of viral load and antibody response in relation to COVID-19 severity

Autopsy findings and venous thromboembolism in patients with COVID-19

Pathophysiology, transmission, diagnosis, and treatment of Coronavirus Disease 2019 (COVID-19): A Review

Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Salivary glands: Potential reservoirs for COVID-19 asymptomatic infection

Age-related rhesus macaque models of COVID-19

The clinical data from 19 critically ill patients with coronavirus disease 2019: a single-centered, retrospective, observational study

Early virus clearance and delayed antibody response in a case of COVID-19 with a history of co-infection with HIV-1 and HCV

A novel coronavirus from patients with pneumonia in China

Left y-axis: stimulation index of IFN-γ, right y-axis: stimulation index of IL-2. Blue and red dotted lines indicate stimulation indexes of 2 in IFN-γ and IL-2, respectively

This work was supported by grants from the Japan Agency for Medical Research and Development (AMED) under Grant Numbers 19fk0108172, 20nk0101615, and