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Major Article the Trend of Neutralizing Antibody Response Against SARS medRxiv preprint doi: https://doi.org/10.1101/2020.08.05.20168682; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 Major Article 2 The Trend of Neutralizing Antibody Response Against SARS-CoV-2 and the 3 Cytokine/Chemokine Release in Patients with Differing Severities of COVID-19: All 4 Individuals Infected with SARS-CoV-2 Obtained Neutralizing Antibody 5 Lidya Handayani Tjan,1* Tatsuya Nagano,2* Koichi Furukawa,1 Mitsuhiro Nishimura,1 Jun Arii,1 6 Sayo Fujinaka,3 Sachiyo Iwata,4 Yoshihiro Nishimura,2 Yasuko Mori1 7 *These authors contribute equally to the work 8 9 1Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School 10 of Medicine, Kobe, Hyogo, Japan 11 2Division of Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate 12 School of Medicine, Kobe, Hyogo, Japan 13 3Clinical Laboratory Department, Hyogo Prefectural Kakogawa Medical Center, Kakogawa, 14 Japan 15 4 Division of Cardiovascular Medicine, Hyogo Prefectural Kakogawa Medical Center, Kakogawa, 16 Japan 17 18 Corresponding author: 19 Yasuko Mori, MD, PhD 20 Division of Clinical Virology, Center for Infectious Diseases 21 Kobe University Graduate School of Medicine 22 7-5-1 Kusunoki-cho, Chuo-ku, Hyogo 650-0017, Japan. 23 Tel: +81-78-382-6272, Fax: +81-78-382-6879 24 E-mail: [email protected] 25 26 Keywords : COVID-19, severe case, neutralizing antibody, cytokine 27 Running title: Neutralizing antibody in COVID-19 28 Summary : 29 Neutralizing antibody against SARS-CoV-2 is induced at high titer in severe COVID-19 30 patients, followed by termination of virus replication while continuously high 1 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.08.05.20168682; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 31 cytokines/chemokines. Convalescent plasma therapy could be administered early to eliminate 32 virus, followed by corticosteroid after viral genome disappearance. 33 2 medRxiv preprint doi: https://doi.org/10.1101/2020.08.05.20168682; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 34 35 ABSTRACT 36 Background: COVID-19 patients show a wide clinical spectrum ranging from mild respiratory 37 symptoms to severe and fatal disease, and older individuals are known to be affected more 38 severely. Neutralizing antibody for viruses is critical for their elimination, and increased 39 cytokine/chemokine levels are thought to be related to COVID-19 severity. However, the trend 40 of the neutralizing antibody production and cytokine/chemokine levels during the clinical course 41 of COVID-19 patients with differing levels of severity has not been established. 42 Methods: We serially collected 45 blood samples from 12 patients with different levels of 43 COVID-19 severity, and investigated the trend of neutralizing antibody production using 44 authentic SARS-CoV-2 and cytokine/chemokine release in the patients' clinical courses. 45 Results: All 12 individuals infected with SARS-CoV-2 had the neutralizing antibody against it, 46 and the antibodies were induced at approx. 4-10 days after the patients' onsets. The antibodies in 47 the critical and severe cases showed high neutralizing activity in all clinical courses. Most 48 cytokine/chemokine levels were clearly high in the critical patients compared to those with 49 milder symptoms. 50 Conclusion: Neutralizing antibodies against SARS-CoV-2 were induced at a high level in the 51 severe COVID-19 patients, indicating that abundant virus replication occurred. 52 Cytokines/chemokines were expressed more in the critical patients, indicating that high 53 productions of cytokines/chemokines have roles in the disease severity. These results may 54 indicate that plasma or neutralizing antibody therapy could be a first-line treatment for older 55 patients to eliminate the virus, and corticosteroid therapy could be effective to suppress the 56 cytokine storm after the viral genome's disappearance. 3 medRxiv preprint doi: https://doi.org/10.1101/2020.08.05.20168682; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 57 INTRODUCTION 58 In December 2019, a new coronavirus was identified as the cause of a pneumonia 59 outbreak in Wuhan, China [1]. The identified virus was related to severe acute respiratory 60 syndrome coronavirus (SARS-CoV), and it was thus named SARS-CoV-2 [2]. Since then, 61 SARS-CoV-2 has spread to almost of the world's countries. According to World Health 62 Organization (WHO) data per 31 July 2020, there were more than 17 million confirmed cases 63 with approx. 650,000 deaths worldwide. 64 The clinical spectrum of COVID-19 varies from mild to severe and fatal infections. The 65 comorbidities related to disease severity include older age, hypertension, diabetes mellitus, 66 obesity, malignancy, and more [3-5]. In addition to the identified comorbidities associated with 67 poorer prognosis in COVID-19, unique immune responses against SARS-CoV-2 infection 68 among different individuals may explain different clinical outcomes observed in infected 69 populations. Patients with a severe infection induce a higher antibody titer compared to those 70 with a mild infection[6], and this is also correlated with neutralizing antibody titer [6, 7] 71 Fatality due to COVID-19 has been reported to be linked to a hyperinflammmatory state 72 characterized by the cytokine release syndrome (CRS) [8]. Among many different types of 73 cytokines and chemokines that have been identified, interleukin (IL)-6 has been consistently 74 reported to reflect the clinical severity of COVID-19 and has been the target of therapy in severe 75 cases [9, 10]. 76 Based on the available studies regarding the immune response against SARS-CoV-2, it is 77 clear that humoral immunity is induced in COVID-19 patients. However, the trend of 78 neutralizing antibody production and cytokine/chemokine levels during the clinical course of 79 COVID-19 in patients with differing levels of disease severity has not been well explored. Here, 4 medRxiv preprint doi: https://doi.org/10.1101/2020.08.05.20168682; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 80 we serially collected blood samples from COVID-19 patients with different disease severities 81 and analyzed the trend of neutralizing antibody production during patients' clinical courses by 82 using authentic SARS-CoV-2 and determining the cytokine/chemokine release. 83 Although recent studies have used a pseudotyped virus for neutralizing assay[11, 12], 84 inconsistent results in neutralizing assay have been shown between pseudotyped and authentic 85 viruses [13-15]. In this study, authentic virus was consistently used to examine the neutralizing 86 activity against SARS-CoV-2. 87 88 PATIENTS AND METHODS 89 Population and samples 90 A total of 12 COVID-19 patients, diagnosed both clinically and based on detection of 91 virus genome by polymerase chain reaction (PCR) were included in this study. All patients were 92 hospitalized at Hyogo Prefectural Kakogawa Medical Center, which is one of the 55 publicly 93 designated medical institutions for infectious diseases in Japan. Blood samples were serially 94 collected from each of the patients. 95 Additionally, seven age-matched healthy adults who were SARS-CoV-2 IgG-negative 96 medical staff from the same facility we described [16] and Kita-harima Medical Center were 97 included in this study as negative controls. IgG was examined using an immunochromatographic 98 test kit (2019-nCoV Ab Test; INNOVITA, Hebei, China) as described [16] 99 100 Serum neutralizing assay against SARS-CoV-2 101 To evaluate neutralizing activity of serum, we performed a neutralization test against 102 SARS-CoV-2 (Biken-2 strain) which was kindly provided from Research Foundation for 5 medRxiv preprint doi: https://doi.org/10.1101/2020.08.05.20168682; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 103 Microbial Diseases of Osaka University (BIKEN), Osaka University. The neutralization test was 104 performed in biosafety level 3 laboratory. For this purpose, 4x104 Vero E6 (TMPRSS2) cells 105 [17] per well were seeded in 96-well tissue culture microplates 24 hr before the assay. Duplicate 106 samples of a three-fold serial dilution of heat-inactivated (56°C, 30 min) serum were prepared 107 using Dulbecco's Modified Eagle’s Medium as the diluent and mixed with 100 tissue culture 108 infectious dose (TCID)50 of virus and incubated at 37°C for 1 hr. After this incubation, the 109 serum-virus mixture was added to Vero E6 (TMPRSS2) cells and incubated at 37°C for 3 days.
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