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¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Liu, L. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Wei, Q. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Lin, Q. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Fang, J. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Wang, H. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Kwok, H. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Tang, H. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Nishiura, K. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Peng, J. in: JCI | PubMed | Google Scholar |

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Tan, Z. in: JCI | PubMed | Google Scholar |

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Wu, T. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Cheung, K. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Chan, K. in: JCI | PubMed | Google Scholar |

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Alvarez, X. in: JCI | PubMed | Google Scholar |

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Qin, C. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Lackner, A. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Perlman, S. in: JCI | PubMed | Google Scholar |

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Yuen, K. in: JCI | PubMed | Google Scholar

¹AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

²HKU-AIDS Institute Shenzhen Research Laboratory and AIDS Clinical Research Laboratory, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Key Clinical Department of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, China.

³Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China.

⁴Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, USA.

⁵Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

⁶State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Address correspondence to: Zhiwei Chen or Li Liu, AIDS Institute and Department of Microbiology, State Key Laboratory of Emerging Infectious Disease, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong S.A.R., China. Phone: 852.28199831; Email: zchenai@hku.hk (ZC). Phone: 852.39179094; Email: liuli71@hku.hk (LL).

Authorship note: AL is deceased.

Find articles by Chen, Z. in: JCI | PubMed | Google Scholar |

Abstract

Newly emerging viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome CoVs (MERS-CoV), and H7N9, cause fatal acute lung injury (ALI) by driving hypercytokinemia and aggressive inflammation through mechanisms that remain elusive. In SARS-CoV/macaque models, we determined that anti–spike IgG (S-IgG), in productively infected lungs, causes severe ALI by skewing inflammation-resolving response. Alveolar macrophages underwent functional polarization in acutely infected macaques, demonstrating simultaneously both proinflammatory and wound-healing characteristics. The presence of S-IgG prior to viral clearance, however, abrogated wound-healing responses and promoted MCP1 and IL-8 production and proinflammatory monocyte/macrophage recruitment and accumulation. Critically, patients who eventually died of SARS (hereafter referred to as deceased patients) displayed similarly accumulated pulmonary proinflammatory, absence of wound-healing macrophages, and faster neutralizing antibody responses. Their sera enhanced SARS-CoV–induced MCP1 and IL-8 production by human monocyte–derived wound-healing macrophages, whereas blockade of FcγR reduced such effects. Our findings reveal a mechanism responsible for virus-mediated ALI, define a pathological consequence of viral specific antibody response, and provide a potential target for treatment of SARS-CoV or other virus-mediated lung injury.

Introduction

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes fatal human respiratory disease (1–3). Patients with SARS (hereafter referred to as SARS patients) displayed the characteristics of acute lung injury (ALI), including diffuse alveolar damage (DAD), epithelial necrosis, and fibrin and hyaline deposition (4, 5). Most patients who die of SARS develop acute respiratory distress syndrome (ARDS), the most severe form of ALI (6, 7). Recent outbreaks of severe acute respiratory infections of emerging viruses, including Middle Eastern respiratory syndrome CoVs (MERS-CoV), highly pathogenic avian influenza viruses (e.g., H5N1 and H7N9), highlight the need to characterize the mechanisms responsible for virus-mediated ALI or ARDS.

Fundamental to ARDS is the acute onset of lung inflammation, which is intimately tied to monocyte/macrophage polarization and function (7, 8). Lung macrophages are highly plastic and heterogeneous cells that are resident in the lung interstitium and alveoli or recruited upon inflammatory stimuli. Inflammatory monocytes and resident tissue macrophages play critical roles in initiating and maintaining inflammation during the acute stage of ARDS, as well as in the resolution phages of inflammation and recovery from ARDS. At steady state, resident macrophages are normally quiescent to prevent damaging the alveoli and are critically involved in normal tissue homeostasis. After tissue injury or during infection, resident macrophages become activated. Circulating monocytes can be efficiently recruited to the site of injury. Inflammatory monocytes/macrophages (IMMs) and resident macrophages undergo marked phenotypic and functional changes, and they can be classified into proinflammatory (M1 or classically activated) and inflammatory-resolving (M2, alternatively activated, wound-healing, or antiinflammatory) macrophages, with a continuum of macrophage polarization existing beyond these discrete categories (9). During acute infection, monocytes/macrophages often display a phenotype of classically activated macrophages. These cells mediate host defenses against viruses and also promote lung injury by producing nitric oxide (NO); ROS; IL-1, IL-6, and IL-8; and TNF. Simultaneously, some macrophages may become alternatively activated, exerting antiinflammatory function and regulating wound healing by producing matrix metalloproteinases (MMPs), growth factors, and antiinflammatory cytokines, particularly TGF-β. When the pathogen or inflammatory stimulus is eliminated, proinflammatory macrophages diminish. The predominant macrophage population assumes a wound-healing phenotype. At the final recovery stage, macrophages show a regulatory/suppressive phenotype, secreting increased levels of IL‑10, which facilitates the resolution of wound healing and restores homeostasis. When the wound-healing response is well organized and controlled, the inflammatory response resolves quickly, and normal tissue architecture is restored. Disturbances in wound-healing response can lead to uncontrolled production of inflammatory mediators, contributing to a state of persistent injury (9–11). In patients who eventually died of SARS (hereafter referred to as deceased patients) and animal models, extensive lung damage is associated with high initial viral loads, increased IMM accumulation in the lungs, and elevated serum proinflammatory cytokine (IL-1, IL-6, IL-8, CXCL-10, and MCP1) levels (4, 5, 12, 13). While much is known about the terminal phase of SARS, little is known about the early immune events during the acute phase of infection. Studies defining macrophage heterogeneity and function during acute infection and ALI using nonhuman primate and patient specimens are limited, and the factors driving the hypercytokinemia and aggressive inflammation remain elusive.

During the SARS outbreak in Hong Kong, most patients (70%–80%) presented with abnormal chest radiographs, with approximately one-quarter of these individuals progressing to ALI. After 12 days, 80% of these SARS patients developed ARDS, coincident with IgG seroconversion (6). In a detailed analysis of antibody responses against SARS-CoV spike (S) glycoprotein, we reported that the anti-S neutralizing antibody (NAb) response developed significantly faster in deceased patients compared with recovered patients after the onset of clinical symptoms (14). It took an average of 20 days for the recovered patients to reach their peak of NAb activities, as opposed to only 14.7 days for the deceased patients. Moreover, the actual NAb titer is significantly higher in deceased patients compared with that in the recovered patients during the same time period (14). These findings suggest a role of anti-S antibodies in SARS-CoV–mediated ALI during acute infection. Consistently, preexisting serum antibodies against influenza antigens were found to associate with worse clinical severity and poor outcomes in patients during the 2009 influenza pandemic (15, 16). Moreover, multiple vaccine platforms and viral infection appeared to induce SARS-CoV–specific immune memory that enhanced lung inflammation following homologous challenge in mice and African green monkeys (17–19). The mechanism responsible for the immunopathologic reaction remains elusive. Recent studies suggested that T cells play a crucial role in protection of mice against lethal SARS-CoV infection (20–22). Enhanced pulmonary immunopathology in vaccinated and challenged animals reflects an inadequate Th1 response (22, 23). The role of virus-specific antibody response in SARS-CoV–induced lung injury has yet to be clearly defined. Therefore, we used vaccination and anti–S-IgG passive immunization strategies to evaluate the effects of anti-S antibodies on SARS-CoV–induced ALI in Chinese rhesus monkeys (Macaca mulatta). Our results now show that, in productively infected lungs, anti–S–IgG causes severe ALI by skewing inflammatory resolving responses during acute infection.

Results

Vaccine-induced S-specific immunity resulted in severe ALI in SARS-CoV infected Chinese macaques. We initially compared the pathological changes in the lungs of rhesus macaques vaccinated with a modified vaccinia Ankara (MVA) virus encoding full-length SARS-CoV S glycoprotein (ADS-MVA) or control at 7 and 35 days after pathogenic SARS-CoV_PUMC (1 × 10⁵ tissue culture infectious dose of 50% [TCID₅₀]) challenge (24). Three healthy macaques were included as controls. Sixteen macaques were i.m. immunized twice: 8 animals with ADS-MVA and another 8 animals with a control MVA (ADC-MVA) (Figure 1A) (24). Vaccination with ADS-MVA induced high levels of anti–SARS-CoV NAbs in all 8 macaques, with sera IC₅₀ values ranging from 10,232- to 28,703-fold dilutions (Figure 1B). None of the macaques developed fever during or after vaccination (normal body temperature 37.8°C–38.1°C). The IC₅₀ values of these sera were maintained, with an over-2,000–fold reciprocal serum dilution at 4 weeks after the secondary immunization, a time when all of the animals were i.n. challenged (Figure 1B). After viral challenge, all macaques developed fever between 1 and 5 days after inoculation (dpi) ranging from 38.6°C–39.2°C, and sera from control vaccinated macaques showed increased neutralizing activity after 14 dpi, indicating establishment of infection (Figure 1B). SARS-CoV RNA was readily detected on oral swabs from all control macaques examined at multiple time points using real-time reverse transcription PCR (real-time RT-PCR) but only in 3 of the 8 ADS-MVA–vaccinated macaques with lower NAb titers at 2 dpi (SL11, SE13, and SL14) (Figure 1C), suggesting reduced productive viral infection in the immunized macaques through S-specific immunity.

Figure 1

ADS-MVA–induced S-specific immune response enhanced pulmonary pathology in SARS-CoV–infected Chinese rhesus macaques. (A) Experimental design used to investigate the influence of S-specific immunity on SARS-CoV–induced lung injury. Two groups of Chinese rhesus macaques (n = 8/group) were subjected to i.m. injections of ADS-MVA or control vaccine ADC-MVA at weeks 0 and 4, followed by i.n. challenge with live pathogenic SARS-CoV_PUMC (1 × 10⁵ TCID₅₀) at 4 weeks after the second vaccination. Four animals each were sacrificed at 1 and 5 weeks after inoculation. Three healthy macaques were included as controls. (B) Serum neutralizing activity. Sera collected from macaques were tested for a capacity to neutralize SARS-CoV pseudotype virus. (C) Detection of viral RNA in oral swabs. SARS-CoV RNA was detected by nested RT-PCR in the swabs at the indicated time points relative to infection. (D) Pathology changes of the lung tissue. Sections were stained with H&E. D shows symptom of acute DAD exhibited in 6 of 8 ADS-MVA–vaccinated macaques with extensive exudation (yellow arrow), hyaline membranes lining the alveolar walls (black arrows), and massive cell infiltration in alveolar cavities (white arrow). Left image shows a low magnification overview (100×). Middle image shows higher magnification of the boxed area in left image (200×). Right image shows minor inflammation observed in 7 macaques received ADC-MVA (n = 8) with slight alveolar septa broadening and sparse monocyte infiltration (original magnification, 100×). (E) Histopathological scores of the ADS-MVA group, including lung samples collected at both 7 and 35 dpi, were compared against the ADC-MVA control group. See Supplemental Figure 1 for the scoring index based on severity of lung histopathology. Data represent mean ± SEM values. Statistical analysis was undertaken using 2-tailed unpaired Student’s t test. *P < 0.05, n = 4. (F) Correlation of lung histopathological scores of all macaques with sera NAb titers at 0 dpi. Solid lines denote the relationship between histopathology scores and serum neutralizing activity. Statistical analysis was performed using Spearman’s rank correlation test.

However, histological examination revealed acute DAD with various degrees of severity in 6 ADS-MVA–vaccinated macaques at 7 and 35 dpi, whereas most control macaques in the ADC-MVA group showed minor to moderate inflammation (Figure 1D). To better characterize the pathological changes, we adopted a 6-grade scoring system to describe the severity of the lung damage from least severe to most severe: 0, –; 1, +; 2, ++; 3, +++; 4, ++++; and 5, +++++ (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.123158DS1). In the control vaccinated group, 5 macaques showed minor inflammation with slight septa broadening and inflammatory cells infiltration that we scored as + (CL23, CE25, CE20, CE21, and CL19; Figure 1D, Supplemental Figure 1B, and Supplemental Figure 2). Two macaques showed moderate inflammation with more interstitial mononuclear inflammatory infiltration and were scored as ++ (CL26, CE24; Supplemental Figure 1C and Supplemental Figure 2). Only 1 macaque showed typical symptoms of acute DAD with extensive exudation and cell infiltration at 35 dpi (CL22, scored as ++++; Supplemental Figure 2). In the ADS-MVA–vaccinated group, 4 macaques showed typical symptom of acute DAD with extensive exudation and many-cell infiltration in alveolar cavities (SL11, SE12, SL16, and SL18, scored as ++++; Supplemental Figure 1E and Supplemental Figure 2). One macaque showed severe acute DAD with exudation, hyaline membrane formation along the alveoli, pneumocyte desquamation, and damaged alveolus filled with hemorrhage and inflammatory cells (SL15, scored as +++++; Figure 1D). One macaque showed early symptom of acute DAD (SL14, scored as +++; Supplemental Figure 1D), and the remaining 2 macaques showed moderate inflammation (SE13 and SE17, scored as ++; Supplemental Figure 2). The comparison of lung histopathlogical scores of 2 groups showed significantly enhanced lung injury in the ADS-MVA–vaccinated group at both 7 and 35 dpi compared with the control ADC-MVA group, suggesting that S-specific — but not MVA-specific — immunity promotes ALI during SARS-CoV infection (Figure 1E). Moreover, there is a moderate correlation between lung pathological scores and sera NAb titers at 0 dpi (Figure 1F), suggesting a role of S-specific antibody in enhancing SARS-CoV–mediated lung injury.

Anti–S-IgG induced severe lung injury during acute SARS-CoV infection. In our previous study, i.n. inoculation of Chinese macaques with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀) led to lower respiratory tract infection in all animals within 2 days (25). However, most of them rapidly cleared infection in the lungs, and all of the animals (n = 8) exhibited mild lung lesions before 3 dpi (25). Severe lung injury in SARS-CoV–infected Chinese macaques has not been detected until 7 dpi (26, 27). To determine the effect of anti-S antibody on the extent of SARS-CoV–mediated lung injury, we adoptively transferred 5 mg (low dose) and 200 mg (high dose) purified anti–S-IgG from ADS-MVA–vaccinated but unchallenged macaques into 2 groups of unvaccinated macaques (n = 6/group) via i.v. injection, and then i.n. challenged recipients with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀) (Figure 2A). As controls, another 2 macaques were administered 200 mg of control IgG (C-IgG) derived from ADC-MVA–vaccinated macaques. We sacrificed half of the macaques in each group at 2 dpi to avoid potential disruption by virus infection–induced antibodies against nucleocapsid protein (N) and other viral proteins — and prior to when induction of ALI was typically observed. The remaining half of the macaques in each group was sacrificed at 21 dpi to evaluate long-term impact (Figure 2A). At day 2, sera from macaques that received low- or high-dose S-IgG showed neutralizing activity, with IC₅₀ values ranging from 10–260 and 1,000–10,000, corresponding to the dose of sera transfer; macaques that received C-IgG failed to show neutralizing activity (Figure 2B and Supplemental Table 1). While NAb titers in the sera of macaques in the high-dose group declined steadily between 2 and 21 dpi, macaques in the low-dose and control groups displayed increased NAb titers over time (Figure 2B), suggesting active viral replication. Indeed, SARS-CoV RNA was detected using real-time RT-PCR on oral swabs, and/or virus was recovered from lung tissue in both control animals and 5 of 6 animals (83%) in the low-dose group. Only 2 of 6 animals (33%) in the high-dose group were viral RNA⁺ (Figure 2C and Supplemental Table 1), suggesting reduced viral production by high-dose S-IgG.

Figure 2

Anti-spike antibodies induced ALI in SARS-CoV–infected Chinese rhesus macaques. (A) Experimental design used to investigate the influence of S-IgG on SARS-CoV–induced lung injury. Two groups of Chinese rhesus macaques (n = 6/group) were subjected to i.v. injection of high-dose (200 mg) or low-dose (5 mg) purified S-IgG from ADS-MVA–vaccinated but unchallenged macaques. As controls, another 2 macaques were administered 200 mg of C-IgG derived from ADC-MVA–vaccinated macaques. After 2 days, 3 groups of macaques were challenged i.n. with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀). Half of the animals from each group were sacrificed at 2 and 21 dpi. (B) Sera from macaques at the indicated time points were tested for the capacity to neutralize SARS-CoV pseudotype virus. (C) Detection of viral RNA in oral swabs. SARS-CoV RNA was detected by nested RT-PCR in the swabs from one of the high-dose S-IgG–treated macaques (n = 6), 3 low-dose S-IgG–treated macaques (n = 6), and 2 C-IgG–treated macaques (n = 2) at the indicated time points. Left y axis shows the viral RNA copy number per milliliter swab. Right y axis shows the serum NAb titers of each macaque at 2 dpi, which are highlighted by shaded area. (D) Pathology changes of the lung tissue (200×). Sections from macaques were stained with H&E. Images show symptom of acute DAD exhibited in macaques received high-dose and low-dose S-IgG (n = 12) with extensive exudation (red arrows), hyaline membranes (black arrows), and massive cell infiltration (yellow arrows) at 2 and 21 dpi. Right panel shows minor and moderate inflammation in the macaques received C-IgG with slight alveolar septa broadening and sparse monocyte infiltration at 2 and 21 dpi. (E) Histopathological scores of the high-dose and low-dose S-IgG groups, including lung samples collected at both 2 and 21 dpi, were compared against the C-IgG group. See Supplemental Figure 1 for the scoring index based on severity of lung histopathology.

Consistent with the findings in the control ADC-MVA–vaccinated group, histopathological examination of the lungs indicated minor and moderate inflammation at day 2 and 21 in C-IgG recipients, respectively. In contrast, all S-IgG recipients exhibited symptoms of acute DAD with various degrees of exudation, hyaline membrane formation, and hemorrhage and inflammatory cells within alveolus at both day 2 and 21 (Figure 2, D and E, and Supplemental Table 2). Of note, despite the presence of high titer sera NAbs during the chronic stage of infection (Figure 1B and Figure 2B), only 1 of 5 C-IgG and control ADC-MVA vaccine recipients showed acute DAD (Figure 1E and Figure 2E), indicating a time-restricted role for S-IgG, which mostly causes severe ALI in acutely infected macaques. Therefore, we conclude that, despite viral suppression, the presence of S-IgG at the acute stage of SARS-CoV infection caused severe ALI that persists until late stages.

S-IgG failed to prevent SARS-CoV lower respiratory tract infection and amplified IMM infiltration and accumulation in the lungs. To determine the potential cause of S-IgG–enhanced ALI, we measured viral infection and monocytes/macrophages infiltration in the lungs and serum cytokine profile at 2 dpi. We previously found that pulmonary infection of Chinese macaques could be rapidly controlled. Viral RNA⁺ cells were detectable in the lungs of only 2 of 4 animals at 2 dpi, although all of them were viral nucleoprotein-positive (NP⁺) in the lungs and the hilar lymph nodes (LNs), where the lymphatics of the lungs drain (25). Therefore, we measured viral RNA⁺ cells by in situ hybridization (ISH), as well as NP signals in the lungs and the hilar LNs by IHC staining to evaluate the actual pulmonary infection of each macaque. Consistent with the results by viral isolation (Supplemental Table 1), viral RNA was detected in pneumocytes of 1 macaque in the high-dose (HS02, 33%), 2 macaques in the low-dose (LS01, LS02; 66%), and the C-IgG–treated macaque (Figure 3A and Supplemental Table 3), suggesting reduced productive infection by S-IgG. However, an NP antibody identified positive signals in the lungs and hilar LNs of all S-IgG and C-IgG recipients (7 of 7 macaques) but not healthy controls (Figure 3B and Supplemental Table 3), suggesting that S-IgG failed to prevent SARS-CoV entry, and SARS-CoV established lower respiratory tract infection in all of the challenged animals.

Figure 3

SARS-CoV infection and monocytes/macrophages infiltration in the lungs of C-IgG– and S-IgG–treated macaques at 2 dpi. (A) Representative images of SARS-CoV RNA⁺ (TRITC) and AE1/AE3⁺ (FITC) cells in the lungs of infected macaques (white arrows). The upper photo shows a low magnification overview (200×); the bottom photo shows the boxed area in the upper photo. (B) Representative images of viral protein (NP) immunostaining of the lungs and hilar lymph nodes (LN) of 7 infected and 3 uninfected animals (TRITC, white arrows) (original magnification, 200×). (C) Representative images of viral NP and monocytes/macrophages. These sections showed significantly increased IMMs in the lungs of the S-IgG group compared with the C-IgG group (MAC387⁺, CD163⁺, or CD68⁺; blue arrows). Tissue samples are double-immunostained for the SARS-CoV NP (TRITC) and markers for macrophages, including MAC387 (FITC), CD163 (FITC), and CD68 (FITC). Within the panel of representative images for the C-IgG and S-IgG groups, the left panel shows a low magnification overview; the right panel shows the boxed area in the left panel (original magnification, 200×).

Consistent with previous findings in SARS-CoV–inoculated naive Chinese macaques (n = 4) (25), double-immunostaining with the NP antibody and antibodies specific to the stages of macrophage differentiation and inflammation (MAC387, CD68, CD163) revealed significant infiltration of IMMs in the lungs of C-IgG recipients, consisting of newly infiltrated (MAC387⁺) and inflammatory (CD163⁺) monocytes/macrophages (Figure 3C and Supplemental Figure 3). The CD163⁺ cells greatly exceeded the CD68⁺ population and typically gathered around NP⁺ cells to form cell clusters (Figure 3C), showing an association between viral infection and IMM infiltration in these macaques.

Compared with C-IgG recipients, more robust signals for MAC387 and CD163 were identified in the lungs of S-IgG recipients (Figure 3C and Supplemental Figure 3), suggesting enhanced IMM infiltration by S-IgG. MAC387⁺ and CD163⁺ monocytes/macrophages not only gathered around NP⁺ cells, but also formed clusters in tissues with little NP signal (Figure 3C), suggesting that infection-induced recruitment from circulation continued after viral clearance and accumulated at the inflamed site. Moreover, more-concentrated CD68⁺ signals were found, indicating altered macrophages functional response by S-IgG treatment (Figure 3C). Consistently, elevated serum levels of IL-8 were observed in macaques treated with high-dose S-IgG at 2 dpi (Supplemental Figure 3), and the IL-8 levels are strongly correlated with anti–SARS-CoV NAb titers (Supplemental Figure 3). IL-8 is a macrophage-derived inflammatory cytokine important in initiating ALI. These results, therefore, suggest that S-IgG likely altered macrophage functional response in the lungs during acute infection, resulted in increase in IL-8 production, and enhanced ALI and IMM infiltration and accumulation.

Alveolar monocytes/macrophages assumed a wound-healing function as early as 2 dpi in macaques not treated with S-IgG. Macrophage activation is dynamic and plastic. Different monocyte/macrophage activation statues have different specialized and critically timed roles, taking part in either the initiation and maintenance or resolution of inflammation (9). A well-organized wound-healing response leads to timely resolution of inflammation, whereas disturbance in wound-healing response results in persistent inflammation and injury. To understand the S-IgG–caused excessive inflammation, we further defined macrophage heterogeneity and activation statues in the lung lesions of C-IgG (n = 1) and S-IgG recipients (n = 3) at 2 dpi. Three healthy macaques and 4 naive Chinese macaques challenged with the same dose of SARS-CoV_PUMC and scarified at 2 dpi from a previous study were included as controls (25).

By combining 5 macrophages markers (MAC387, CD68, CD163, HAM56, and CD206) (28–30), 2 subpopulations of resident macrophages were shown in the lungs of Chinese rhesus macaques at steady state as previously described (29). They are interstitial macrophages (IMs; MAC387⁺HAM56^–CD206^–) (Figure 4A) and resident alveolar macrophages (AMs; CD68⁺ HAM56^–) expressing low levels of CD163 and CD206, a marker specific to the macrophage wound-healing response, corresponding to their intrinsic antiinflammatory function (Figure 4B) (29). In lung lesions of both C-IgG–treated and naive macaque groups challenged with SARS-CoV_PUMC, IMMs were further characterized and divided into 2 subpopulations. One subpopulation was the recently recruited MAC387⁺HAM56^–CD68^– monocytes (Figure 4C) (29), which is like IM but is larger in size and primarily coexpressed CD163 (Supplemental Figure 4). The other subpopulation was single CD163⁺ inflammatory macrophages that had differentiated from newly recruited MAC387⁺ monocytes/macrophages (Figure 4D and Supplemental Figure 4) (29). Importantly, resident AMs (CD68⁺) differentiated into HAM56⁺ cells, with a slight increase in numbers and increased size, with stronger CD163 and — more importantly — CD206 staining, suggesting that they are alternatively activated (Figure 4D). Strikingly, elevated levels of TGF-β expression were detected in all MAC387⁺ — and most enlarged CD163⁺ — as well as CD68⁺ macrophages in the lungs of both C-IgG–treated and unvaccinated macaques compared with healthy controls (Figure 4, G–I), indicating that many IMMs and activated resident AMs have assumed a wound-healing function in infected lungs within 2 days of infection.

Figure 4

Comparison of monocyte/macrophage phenotype and function in the lungs of S-IgG– and C-IgG–treated macaques. (A–F) Phenotype of monocytes/macrophages subpopulations in the lungs of healthy, C-IgG–, and S-IgG–treated macaques (original magnification, 200×). These sections were triple-immunostained with antibodies for CD206 (cyan), HAM56 (FITC), and MAC387 (TRITC) (A, C, and E); CD206 (cyan), HAM56 (FITC) (B, D, and F), and CD68 (TRITC) (upper panel in B, D, and F); or CD163 (TRITC) (lower panel in B, D, and F). A and B show 2 subpopulations at steady states, including interstitial macrophages (IM) (MAC387⁺HAM56^–CD206^–) (yellow arrow), and resident alveolar macrophages (AM) (CD68⁺CD163^loCD206^loHAM56^–) (white arrows). C and D show the presence of alternatively activated resident AM and accumulated IMMs in the C-IgG group. Alternatively activated AMs are CD206^hiCD68⁺CD163^hiHAM56⁺ (D, white arrows). IM are MAC387⁺HAM56^–CD206^– (C, yellow arrow). IMMs include newly infiltrating monocytes/macrophages (MAC387⁺) (C, green arrow); and inflammatory macrophages (CD163⁺CD68^–CD206^–HAM56^–) (D, green arrow). E and F show significantly increased number of IMMs and decreased number of alternatively activated macrophages in the S-IgG group. IMMs include newly infiltrating monocytes/macrophages (MAC387⁺) (E, green arrow) and inflammatory macrophages that are CD68⁺HAM56^– (green arrow, F, upper panel) and CD163⁺ CD206^–HAM56^– (green arrow, F, lower panel). Resident AMs are no longer expressing CD206 (CD68⁺HAM56⁺CD163⁺CD206^–) (blue arrow, F, upper panel). (G–J) TGF-β and IL-6 expression in macrophages in the lungs. These sections were double-immunostained with antibodies for TGF-β or IL-6 (TRITC) and MAC387, CD163, or CD68 (FITC). G shows low expression levels of TGF-β and IL-6 in IMs (MAC387⁺) and AMs (CD68⁺) in healthy macaques. H shows increased TGF-β expression in the C-IgG group (white arrows) but increased IL-6 in the S-IgG group (blue arrows). I and J show the numbers of TGF-β⁺ and IL-6⁺ monocytes/macrophages in 3 groups in a 200× field.

S-IgG treatment skewed wound-healing macrophage response in the lungs during acute SARS-CoV infection. Compared with the macaques not treated with S-IgG, the number of IMMs (MAC387⁺ and CD163⁺HAM56^–) significantly increased in S-IgG recipients (Figure 4, E and F). Moreover, many recently recruited MAC387⁺HAM56^– monocytes/macrophages did not coexpress CD163 (Supplemental Figure 4), representing the newest recruited blood monocytes, as a result of enhanced monocytes infiltration by S-IgG. Importantly, the majority of CD163⁺HAM56^– IMMs coexpressed CD68 (Supplemental Figure 4), suggesting a change of function of this subpopulation by S-IgG. Moreover, resident macrophages (CD68⁺HAM56⁺) in the lungs of S-IgG recipients no longer express CD206, which — being surrounded by many CD163⁺ IMMs (Figure 4F) — suggests a loss of wound-healing function and a role in recruiting blood monocytes. Consistently, S-IgG–treated macaques displayed a loss of TGF-β and high expression of IL-6 in all macrophage subpopulations (Figure 4, G, H, and J). IL-6 favors macrophage accumulation at the site of injury and is important for the development of persistent inflammation and ALI (31, 32). Therefore, we conclude that, while AMs underwent phenotypic and functional changes in acutely infected lungs, many monocytes/macrophages assumed a wound-healing function for inflammation resolution, but the presence of S-IgG skewed the wound-healing response, leading to uncontrolled inflammation and tissue injury.

Onset of an antibody response prior to viral clearance is associated with abrogated wound healing responses and increased IMM lung infiltration in unvaccinated Chinese rhesus macaques. To understand the mechanism underlying the time-restricted role of S-IgG in promoting ALI, we conducted a temporal analysis of the productive viral infection, antibody response, and macrophage functional changes in unvaccinated Chinese macaques during the first week of infection. Naive macaques were challenged i.n. with 1 × 10⁵ TCID₅₀ SARS-CoV_PUMC and sacrificed at 2, 3, and 7 dpi (4 macaques/group) (Figure 5A) (25). Successful lower respiratory tract infection was confirmed by detection of viral NP using IHC staining in the lungs of all macaques (25). Viral RNA⁺ alveolar pneumocytes were primarily found at 2 dpi (2 of 4 macaques), and infrequently at 3 dpi (0 of 4 macaques; Figure 5B and Supplemental Table 4) (25