key: cord-0991414-ppo5rv67 authors: Lin, Lan; Luo, Shanshan; Qin, Renjie; Yang, Mengling; Wang, Xiaobei; Yang, Qianqian; Zhang, Yang; Wang, Quansheng; Zhu, Rui; Fan, Heng; Wang, Haijun; Hu, Yu; Wang, Lin; Hu, Desheng title: Long-term infection of SARS-CoV-2 changed the body's immune status date: 2020-07-11 journal: Clin Immunol DOI: 10.1016/j.clim.2020.108524 sha: 47779ca715159735de6c081cf676aef09b31d09e doc_id: 991414 cord_uid: ppo5rv67 The outbreak of SARS-CoV-2-associated pneumonia, a disease called COVID-19, has caused a pandemic worldwide. To investigate the immune responses after infection of SARS-CoV-2 in non-critical patients may help to better understand the disease progression. We collected 334 confirmed COVID-19 cases including 212 still in hospital with nucleic acid test positive on halfway for SARS-CoV-2 and 122 discharged from hospital, compared specific antibodies, immune cells, and cytokine changes between the hospitalized and discharged patients. The hospitalized patients had a longer illness time compared with discharged patients. Analysis of viral loads explained long-term or persistent infection of SARS-CoV-2, which existed with the median time of 18.5 days of the positive nucleic acid test. Serum analysis showed that the specific anti-N IgG antibody was positive in all detected patients after infection of two weeks. Neutrophils, Monocytes, NK cells, and CD4(+) T cells significantly increased, while total lymphocytes and CD8(+) T cells decreased from non-critical hospitalized patients after longer-term infection. Further analysis of the cytokines showed that IL-6, TNF-α, IFN-γ, IL-2, IL-4, and IL-10 from the hospitalized patients were significantly higher, indicating a potential of the increased CD4(+) T cell differentiation. The outbreak of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) -associated pneumonia, a disease called COVID-19 (Coronavirus Disease 2019 ), had caused a pandemic worldwide (1, 2) . Although the fatality rate of COVID-19 is lower, the virus has already caused more death than the other lethal cousin MERS-CoV (Middle East Respiratory Syndrome Coronavirus) and SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) outbreaks combined. As an infectious disease, the pathogen, a betacoronaviruse, now called SARS-CoV-2 has been identified and the whole genome sequencing was reported and disclosure successively (3) . According to the analysis of the structure of spike protein and knockout strains of cell receptor-specific gene, the invading receptor was identified as ACE2 (Angiotensin-Converting Enzyme 2), the same with SARS-CoV (4, 5) . As for clinical classification, most cases were classified as mild with non-pneumonia and mild pneumonia, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious diseases. We extracted demographic data, medical history, symptoms, signs, laboratory findings, and the treatment measures from electronic medical records. Laboratory confirmation of SARS-CoV-2 was done in different hospitals with valid evidence and steroid treatment cases had been excluded from our collected cases. Followed the newly classification methods given by china CDC, mild patients are including non-pneumonia and mild pneumonia, severe are involving serious pneumonia with dyspnea, respiratory frequency ≥30/min, blood oxygen saturation ≤93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio <300, and/ or lung infiltrates >50% within 24 to 48 hours and shortness of breath, the agency calls "critical" patients are with respiratory failure, septic shock, and/or multiple organ dysfunction or failure. (6) The collected cases were divided by whether the patients were still discharged from the hospital, and the data were analyzed according to the following flowchart. J o u r n a l P r e -p r o o f Throat-swab specimens from the upper respiratory tract that were obtained from all patients at admission were maintained in a viral-transport medium. In particular, to ensure the accuracy of the analysis results, only the viral load data acquiring under the same kit and operating conditions were accepted. Categorical variables were expressed as frequency rates and percentages (%), and continuous variables were expressed as mean (SD) if they are normally distributed or median (IQR) if not. Means for continuous variables were compared using independent group t-test when the data were normally distributed; otherwise, the Mann-Whitney test was used. Proportions for categorical variables were compared using the χ2 test, although the Fisher exact test was used when the data were limited. All statistical analyses were performed using SPSS (version 13.0). In all collected cases the patients' median age was 62 years (IQR (interquartile range), 50-68), and the younger were more likely to discharge from the hospital (Table 1) . Of the 334 The majority of the confirmed patients were mild in enrolled cases (Table 1) . We collected the detailed data of the discharged patients of the disease duration, the undetectable time-point of viral nucleic acid, and the illness time of the inpatients (Fig.1A) . The average disease duration from initial symptoms to discharged from hospital was 25.9 days, median time was 25 (IQR, 20-31). And the median time of undetectable time-point of viral nucleic acid was 19 (IQR,13-24.5) (Fig.1B) . The illness time is much longer than the viral undetectable time-point. It suggested that the immunopathological damage still happened in the patients infected with SARS-CoV-2, similar to the SARS-CoV infected patients (15) . Moreover, the time of illness of hospitalized patients was longer than the discharged patients. It also seemed reasonable for most of the simultaneous admission under the special quarantine condition in China with few new infection cases occurring. non-critical patients. The executing discharge rules promoted us to follow up on the inpatient's nucleic acid tests. While it was easy to find that there were still many patients classified as mild ones who couldn't be discharged from hospital. We collected all positive nucleic acid tests in inpatients These suggested the possibility of long-term carrying viruses in both mild and severe cases. It could be speculated that the virus could cause a long-term persistent infection in these nucleic acid positive inpatients with apparent pneumonia but not critical, confirmed by chest CT scans, long-term infection of SARS-CoV-2 occurred in hospitalized non-critical patients. (Fig.3C) . Also, little difference was seen in the discharged (n=8) and hospitalized patients (n=127) (Fig.3D) . Additionally, the same batch of antibody detection was less performed in the discharged patients, thus the discharged patients had less antibody detection J o u r n a l P r e -p r o o f Journal Pre-proof numbers than the hospitalized patients. In conclusion, whatever, these results illustrated that the virus could arouse the acquired immune response of the infected bodies to produce specific antibodies both in inpatients and discharged patients. Specific SARS-CoV-2 antibodies were produced for two weeks of onset. The production of the specific antibodies provided the clues about specific immune responses to SARS-CoV-2, and ample evidence had illustrated the decreased CD8 + T cells counts in critical patients, indicating the influence on cellular immunity. Collected data from blood routine and flow cytometry examinations of peripheral blood help us analyze the immune cell changes after longer-infection of SARS-CoV-2. Based on the results of these two examinations, we found the long-term infection of SARS-CoV-2 increased the account of monocytes, neutrophils, NK cells, and CD4+ T cells, but also declined the total lymphocytes and CD8 + T cells in the hospitalized patients ( Fig.4 , Table 2 ). Lymphocytopenia is one of the most prominent markers of COVID-19, it's also one of the diagnostic criteria for COVID-19 in China. And we found in longer infection inpatients lymphocytopenia deteriorated further. But the increases of the NK cells, neutrophils, and CD4 + T cells in the non-critical patients were much different from the characteristics of severe or critical patients. It might imply the differences between the non-critical or critical patients. The counts of CD4 + T cells was increased, as well as the ratio of CD4 + T cells against CD8 + T cells in the hospitalized non-critical patients, with the unchanged total CD3 + T cells (Fig.4B, 4C ). It should be noted that CD4 + T cells elevation was also present in discharged patients, but the increased proportion was not as obvious as in the patients with long-term but non-critical infection. The J o u r n a l P r e -p r o o f data also showed that the proportion of the increased and out of normal range CD4 + T cells were respectively 43.56% (n=191) and 26.55% (n=113) in the inpatients and discharged patients according to the clinical examining reports. Anyway, all these results revealed that the long-term SARS-CoV-2 infection caused changes in the immune responses in non-critical patients, different from the critical patients. patients. The secretion of cytokines could also reflect the body's immunity to viruses and they play important roles in the regulation of immune responses. We were also very curious about the changes in the body's cytokines secretion after a longer-term infection with SARS-CoV-2. The tests of the cytokines containing IL-6, TNF-α, IFN-γ, IL-2, IL-4, and IL-10 from the hospitalized and discharged patients give evidence. The results showed that after the longer-infection of SARS-CoV-2, all of the cytokines was upregulated (Fig.5 , Table 2 ). It suggested compared with the discharged patients, in hospitalized patients, the body's immune system could present a significantly different immune status for that the different cytokines have different sources and different functions. the inpatients who were still with supporting treatment. As widely acknowledged, the immune system plays an important role in clearing the virus and the adaptive immune protects humans from re-infection, thus, it was important to figure these changes out. In this retrospective study, to investigate the difference of immune responses we analyzed the changes of antibodies, immune cells, and cytokines in hospitalized with positive nucleic acid test and discharged with negative nucleic acid test patients. For the cases of the hospitalized and discharged non-critical cases, we found that for N protein showed no significant difference between the discharged patients and hospitalized patients. And we could not observe the antibodies rise in our study, the probable reason was that the titer of antibody might have reached a platform stage, for the most illness time of these patients were over two weeks in the same batch detection. However, we observed that the persistent infection of the virus in non-critical patients caused the exacerbated lymphopenia and decrease of CD8 + T cells, as well as in the critical patients (19) . And more and more evidence supported that the exhaustion might be In the end, we must emphasize the persistent infection of SARS-CoV2 in non-critical patients and it must attract enough attention. In conclusion, in this study we portrayed the long-term Means for continuous variables were compared using independent group t-test when the data were normally distributed; otherwise, the Mann-Whitney test was used. Comparison of anti-N IgG value between discharged (n=8) and hospitalized patients (n=127). Means for continuous variables were compared using independent group t-test when the data were normally distributed; otherwise, the Mann-Whitney test was used. J o u r n a l P r e -p r o o f Clinical features of patients infected with 2019 novel coronavirus in Wuhan A novel coronavirus outbreak of global health concern A pneumonia outbreak associated with a new coronavirus of probable bat origin Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Sensitivity of Chest CT for COVID-19: Comparison to RT-PCR SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study The use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (COVID-19): The Perspectives of clinical immunologists from China Smoking Upregulates Angiotensin-Converting Enzyme-2 Receptor: A Potential Adhesion Site for Novel Coronavirus SARS-CoV-2 Dysregulation of immune response in patients with COVID-19 in Wuhan, China SnapShot: T Cell Exhaustion Molecular and cellular insights into T cell exhaustion Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients Janeway's immunobiology. New York: Garland science Plasticity of CD4+ T cell lineage differentiation  Interleukin-10 (pg/mL) Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College This study was funded by the grants from the project of Thousand Youth Talents for DH; and