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18 Aug 2020

[Press Release] HKU-Pasteur Discovered That ORF8 And ORF3b Are Major Targets Of SARS-CoV-2 Humoral Immune Response

Press Release 

HKUMed researchers discovered that ORF8 and ORF3b are major targets of SARS-CoV-2 humoral immune response and can be used for more specific testing of COVID-19 infection
 
Researchers from HKU-Pasteur Research Pole at the School of Public Health, LKS Faculty of Medicine of The University of Hong Kong (HKUMed) used Luciferase Immunoprecipitation System (LIPS) assay and discovered that ORF8 and ORF3b are major targets of SARS-CoV-2 humoral immune response and can be used for more specific testing of COVID-19 infection. The study is the first among research teams worldwide to determine the most comprehensive “landscape” of antibody responses to SARS-CoV-2. The ground-breaking findings on COVID-19 serology are now published in Nature Immunology, a leading scientific journal. 
 
 
Antibody testing is an important tool in the response to COVID-19. It allows us to define the true extent of infection in the community, may help to confirm diagnosis in suspected patients and is needed for evaluation of vaccines. To date, SARS-CoV-2 antibody testing has been focussed on two proteins of the virus - the Spike protein as it correlates with neutralising capacity of the serum,  and the Nucleocapsid protein as it is abundantly made by the virus and thus more likely to elicit detectable responses. Both proteins have potential problems with false positive reactions due to cross reactivity with antibody to other human coronaviruses which are common in the population. 
 
The research team used the Luciferase Immunoprecipitation System (LIPS) assay, an antigen discovery tool previously used for screening cancer and new pathogen antigens, and assessed the antibody responses to 15 different SARS-CoV-2 proteins in COVID-19 patients. They identified that two non-structural proteins, ORF8 and ORF3b, are key SARS-CoV-2 targets that elicit early and potent antibody responses in COVID-19 patients. These proteins are unique to SARS-CoV-2, thus are able to avoid the problem of cross-reactivity with other “common cold” human coronaviruses. Such robust antibody responses against these two viral proteins could be consistently detected in the majority of COVID-19 patients (96.5% sensitivity). 
 
Currently, ORF3b and ORF8 proteins are not mainstream targets for COVID-19 diagnosis. These findings will improve the serological tests for COVID-19 and enhance the development of second-generation diagnostic tests.
 
Figure generated by Biorender
Researchers at HKU Pasteur Research Pole, School of Public Health, LKS Faculty of Medicine at The University of Hong Kong (HKUMed) used Luciferase Immunoprecipitation System (LIPS) assay and discovered that ORF8 and ORF3b are major targets of SARS-CoV-2 humoral immune response and can be used for more specific testing of COVID-19 infection. 
 
Blood from COVID-19 patients contains antibodies early after infection, that bind ORF3b and ORF8 proteins. These antibodies were detected by proteins tagged with fluorescent labels by LIPS. As these proteins are unique to the SARS-CoV-2 virus, antibodies from uninfected people would not bind these targets. Thus, LIPS is able to demonstrate a significant difference between COVID-19 patients and uninfected (negative) people.
 
 
 
About the research team:
The research was conducted by a team led by Dr Sophie Valkenburg, Research Assistant Professor, HKU-Pasteur Research Pole (HKU-PRP), School of Public Health, HKUMed. Other members of the research team from HKU-PRP included Ms Asmaa Hachim, Research Assistant; Ms Carolyn A Cohen, PhD student; and Dr Niloufar Kavian-Tessler, Post-doctoral Fellow and Honorary Visiting Fellow from Paris Descartes University and Cochin Hospital (France). Members from the School of Public Health, HKUMed included Dr Alex Chin Wing-hong, Investigator; Dr Daniel Chu Ka-wing, Research Assistant Professor; Dr Chris Mok Ka-pun, Research Assistant Professor; Dr Ranawaka APM Perera, Research Assistant Professor; Professor Leo Poon Lit-man, Professor and Head of Division of Public Health Laboratory Sciences; and Professor Malik Peiris, Tam Wah-Ching Professor in Medical Science and Chair Professor of Virology. Members from Princess Margaret Hospital were Dr Owen TY Tsang, Medical Director, Hospital Authority Infectious Disease Centre; and Dr Yiu Cheong Yeung, Specialist in Respiratory Medicine, Department of Medicine & Geriatrics. 
 
This project was supported by a Commissioned Grant from the Health and Medical Research Fund (Ref: COVID-190115), Food and Health Bureau, and General Research Fund (Ref: 17113718), Government of Hong Kong Special Administrative Region.
 
 
About HKU Pasteur Research Pole
HKU-Pasteur Research Pole (HKU-PRP) is a joint laboratory of HKU and Institut Pasteur, co-directed by Professor Roberto Bruzzone and Professor Leo Poon Lit-man, established in 2000 with the aim to developing programmes of excellence in research and education that will generate biological knowledge and advance the understanding and treatment of infectious diseases. HKU-PRP benefits from the outstanding scientific environment offered by the School of Public Health of the LKS Faculty of Medicine, with its significant contributions, both locally and internationally, to research on emerging viral diseases and improving health. Moreover, HKU-PRP is part of the Institut Pasteur International Network, a unique model for health cooperation to further science, medicine and public health with more than 100 years of history.
 
About the School of Public Health, HKUMed 
The School of Public Health, LKS Faculty of Medicine of The University of Hong Kong has a long and distinguished history in public health education and high impact research. With world leading research in infectious diseases as well as on non-communicable diseases of both local and global importance, the School has made significant contributions through its research and advocacy to improve the health of populations and individuals, both locally and globally. The School is a leading research and teaching hub in public health on influenza and other emerging viruses, control of non-communicable and infectious diseases, tobacco control, air pollution, psycho-oncology, behavioural sciences, exercise science, life-course epidemiology, and health economics, health services planning and management. This work has informed international (e.g. the US Food and Drug Administration, Health Canada, the World Health Organization), national and local public health policies.
 
For the latest update from HKUMed on COVID-19:
 
Media enquiries
School of Public Health, HKUMed 
Dr Sophie Valkenburg (Tel: 2831 5501| Email: sophiev@hku.hk)
Dr Niloufar Kavian-Tessler (Tel: 2831 5494 | Email: niloufar@hku.hk)
 
HKUMed
Christiana Ho (Tel: 3917 9535 | Email: hoyan913@hku.hk
Alex Chan (Tel: 3917 9784 | Email: alexisk@hku.hk)
 
 

17 Aug 2020

Systems Biology Approach To Determine Host Immune Responses To COVID-19

Chris Mok and his team keep their relentless efforts in understanding the mechanisms of SARS-CoV-2 and human immune response in this recent paper published in Science with Bali Pulendran from Stanford University's Institute for Immunity:

>>> Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans

The recent emergence of the SARS-coronavirus 2 (SARS-CoV- 2) in Wuhan, China, in December 2019, and its rapid international spread poses a global pandemic. Research has moved rapidly in isolating, sequencing and cloning the virus, developing diagnostic kits, and testing candidate vaccines. However, major questions remain about the dynamic interaction between the human immune system and the SARS-CoV-2 virus and the immunological mechanisms underlying COVID-19 severity.
 
Epidemiological data so far suggest that COVID-19 has a case fatality rate several times greater than that of seasonal influenza. The elderly and individuals with underlying medical comorbidities such as cardiovascular disease, diabetes mellitus, chronic lung disease, chronic kidney disease, obesity, hypertension or cancer have a much higher mortality rate than healthy young adults. The underlying causes are unknown, but may be due to an impaired interferon response, and dysregulated inflammatory responses as observed with other zoonotic coronavirus infections such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS).
 
In this publication, the authors used a systems biological approach (mass cytometry and single cell transcriptomics of leukocytes, transcriptomics of bulk PBMCs, and multiplex analysis of cytokines in plasma), to analyze immune response in 76 COVID-19 patients and 69 age- and sex-matched controls, from two geographically distant cohorts (Hongkong and Atlanta).
 
Interestingly, in PBMCs of COVID-19 patients, there was reduced expression of HLA-DR and pro-inflammatory cytokines by myeloid cells, and impaired mTOR-signaling and IFN-α production by plasmacytoid DCs. In contrast, there were enhanced plasma levels of inflammatory mediators, including EN-RAGE, TNFSF14, and oncostatin-M, which correlated with disease severity and increased bacterial products in human plasma. 
 
Moreover, single-cell transcriptomics results revealed no type-I IFN, reduced HLA-DR in myeloid cells of severe patients, and transient expression of IFN-stimulated genes. This was consistent with bulk PBMC transcriptomics, and transient, low plasma IFN-α levels during infection. 
 
In summary, these results suggest that SARS-CoV-2 infection results in a spatial dichotomy in the innate immune response, characterized by suppression of peripheral innate immunity, in the face of proinflammatory responses reported in the lung. 
 

03 Aug 2020

Lessons Learned From The SARS Pandemic, A Review On The Topic By Prof. Malik Peiris And Prof. Leo Poon

In a new publication, Prof. Malik Peiris, HKU School of Public Health and former co-director the HKU-Pasteur Research Pole, and his successor Prof. Leo Poon, reflect on what the SARS 2003 pandemic in Hong Kong have enabled scientists to understand about the cause of these types of coronaviruses.
 
Severe acute respiratory syndrome (SARS) was the first major global public health crisis of the 21st century. In March 2003, scientists reported to the World Health Organization (WHO) the discovery of a novel coronavirus (CoV) responsible for this newly emerged disease. SARS first emerged in Guangdong, China in November 2002, leading to major outbreaks in the Provincial capital Guangzhou in January. HKU-Pasteur first heard about these outbreaks in mid-February 2003. Hong Kong set up enhanced surveillance for all patients with severe pneumonia of unknown aetiology in Hong Kong, especially those with a travel history to Guangdong. 
 
The two researchers’ first investigation for known respiratory pathogens proved negative. They then started to look more broadly for unusual viruses, including attempting virus culture using a range of cell lines not normally used for respiratory viruses, broad-range PCR/RT-PCR, random primer RT-PCR methods as well as electron microscopy of a lung biopsy from a suspected patient. By March 17, 2003, they began to see subtle changes in FRhk 4 monkey kidney cells inoculated with specimens from two suspected patients. By electron microscopy, they could see virus particles within these cells and we then used fixed infected cells to demonstrate antibody responses in paired sera from a number of suspected SARS patients but not in controls. Using random RT-PCR, they were able to identify the virus as a novel coronavirus. The initial short RNA sequence of 646 nucleotides obtained from the random RT-PCR rapidly allowed the development of RT-PCR assays for detecting SARS patients. 
 
All these findings were shared in real-time via daily teleconferences organized by the WHO to link up laboratories working on this outbreak. Two other laboratories within the network (Centers for Disease Control and Prevention, USA and Bernhard Nocht Institute for Tropical Medicine, Hamburg) reported similar findings from other SARS patients but others were still arguing for other aetiologies (e.g., human metapneumovirus). Sharing of data within the WHO network allowed a rapid consensus that the novel coronavirus was indeed the cause of SARS.
 
 

31 Jul 2020

Institut Pasteur Transverse Research Programs: HKU-Pasteur To Work On The Relationship Between Stem Cells And Infectious Diseases

Flu is a contagious infectious disease caused by the influenza virus. Influenza A and B viruses circulate and cause seasonal epidemics. Each year about 3-5 million individuals develop a severe illness, causing 290 000 to 600 000 deaths. Flu is an infection of the respiratory tract characterized by a sudden onset of symptoms such as fever, cough, headache, muscle and joint pain. Most people recover within a week, but influenza can cause severe illness or death especially in high risk population, such as pregnant women, elderly, young children, individual with chronic diseases and health care workers. 
 
Decline of independence after hospitalization has been reported in the elderly, as well as in specialized residence. This poses a number of issues, including an ever-increasing cost of national health care budgets. So far, researchers are mainly focused on respiratory complications leading to death, but few studies are addressing the causes and the consequences of the associated symptoms
 
Between 2015 and 2018, Barbara Gayraud-Morel, from the unit of Stem Cells and Development (Institut Pasteur Paris) directed by Shahragim Tajbakhsh, had the opportunity to establish a fruitful collaboration with HKU-Pasteur, laying the foundations for this project under the Institut Pasteur Transverse Research Programs.
 
This partnership will unite their expertise on skeletal muscle research, stem cell biology and infectious diseases to investigate the consequences of influenza viral infection at a site where the biology of tissue stem cells has been investigated, notably in skeletal muscles. The observation that inflammatory molecules reach skeletal muscles led them to investigate the stem cell or pathogen driven systemic inflammation interactions which has so far remained unexplored. The compelling preliminary data gathered by the team led them to find that skeletal muscle stem cells respond to the systemic inflammation when there is an intranasal infection by the influenza virus. This opens a new field of research that exposes physiological response of tissues and organs to pathogens.
 
The characterisation of the molecular basis and functionality of this phenomenon will have a significant impact on human health by revealing an appreciated role of stem cells in symptoms associated with systemic inflammation during and after infection. This new field of investigation will inevitably be extended to other tissues and organs, for example, to evaluate if nerve, skin or intestinal stem cells are affected by systemic inflammation and result in altered function. With this new project, the team aims to understand the signalling molecules and mechanisms acting on muscle stem cells remotely from the site of infection, through the characterization of the biological response adopted by the muscle stem cells in this context of viral inflammation. 
 
They will also explore if their observations on muscle stem cells in influenza-mediated inflammation could be a landmark of a more general stem cell response to acute inflammation in other contexts. To this end, this project associates the Centre for Translational Science Unit of Fabrice Chrétien (Institut Pasteur Paris), which has the expertise in another distinct systemic infection model, sepsis, in order to explore and compare the two paradigms, one related to viral response, and the other, a polybacterial infection. 
 
This project, which is the result of synergistic expertise within the Pasteur network, opens a new field of investigation on the relationship between stem cells and infectious diseases. Strong preliminary data have paved the way for us to explore this area further and position this topic at the forefront of international research. 
 

29 Jul 2020

COVID-19: Even Mild Cases Develop A Robust, Neutralizing Antibody Response

Chris Mok and his team worked on the development of the immune response in COVID-19 patients in collaboration with Dr Jincun Zhao from the State Key Laboratory of Respiratory Disease of Guangzhou Medical University. During their research, they found that even mild cases have a robust, neutralizing antibody response.

As of late July, the World Health Organization (WHO) has reported more than 15 Million cases of COVID-19 worldwide, including more than 640,000 deaths, and still little is known about the kinetics, tissue distribution, cross-reactivity and neutralization antibody response to SARS-COV-2.

In this publication, in order to monitor viral shedding and antibody responses in patients with severe and mild disease in different tissues, the authors recruited two groups of RT-PCR confirmed COVID-19 patients, including 12 severe patients in ICUs who needed mechanical ventilation and 11 mild patients in isolation wards.

They found that COVID-19 patients with different severity of disease showed different patterns of viral shedding and antibody responses. Severe patients had more prolonged viral shedding in a variety of tissues than mildly ill patients. SARS-CoV-2-specific antibodies were found in tissues outside the respiratory tract in severe patients. Detection of antibody responses in urine and other body fluids could be used as a marker to determine disease severity. 

Interestingly, by using plasma from SARS, MERS and COVID-19 patients, strong cross-reactivities were detected between SARS-CoV-2 and SARS-CoV, but not MERS-CoV which is an important information for differential diagnosis in Middle East countries. 

More importantly, antibodies against N or S protein were correlated with neutralizing antibody titers which may be useful when screening convalescent plasma for passive transfusion therapy.

In summary, this study provides comprehensive information on kinetics, tissue distribution, cross-reactivities and neutralization of antibody responses in COVID-19 patients, and will improve our understanding of humoral immune response in human after SARS-CoV-2 infection as well as shedding light on diagnosis, prognosis, convalescent plasma transfusion therapy and epidemiology studies of SARS-CoV-2 infection in human.

Their findings are published by the Journal of Clinical Investigation:

>>> Kinetics Of Viral Load And AntibodyResponse In Relation To COVID-19 Severity

21 Jul 2020

Chris Mok’s Collaborative Project With Institut Pasteur Tunis Selected By The Institut Pasteur International Covid-19 Task Force

Since January 2020, the Institut Pasteur Covid-19 Task Force and the Institut Pasteur International Network have been extremely active worldwide in responding with science to the pandemic caused by the SARS-CoV-2 coronavirus. 

The joint project developed by Chris Mok’s team at the HKU-Pasteur Research Pole and by the Institut Pasteur Tunis, Tunisia, will be funded by the Covid-19 Task Force as part of the second round of projects selection. 

In this framework, the project “EASI: ELISA Assays development for SARS-COV2” will focus on the development of diagnostic tools. “ELISA” is a widely established technology to detect the presence of antigens in samples.

Screening with this ELISA assay is a reliable approach for large-scale sero-epidemiological studies, which are crucial to assess infection attack rates in the population and to accurately define disease severity and herd immunity.

Scientific collaboration in times of global pandemic is crucial to capitalize on various countries and laboratories’ expertise in order to develop groundbreaking projects. 

 

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