Major Article the Trend of Neutralizing Antibody Response Against SARS

Home , Neutralizing antibody

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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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1 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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.

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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.

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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,

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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.

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]

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

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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.

110 Neutralizing antibody titer was determined as the highest serum dilution that did not show any

111 cytopathic effects by observation under microscope. These experiments were repeated three

112 times independently.

113

114 Chemokine measurements

115 A total of 48 cytokines, chemokines, and growth factors were measured in serum samples

116 using the Bio-Plex Pro Human Cytokine Screening 48-plex panel (Bio-Rad, Hercules, CA), and

117 results were read using the Bio-Plex 200 system following the manufacturer's instructions. The

118 cytokines, chemokines, and growth factors detected in this system are listed in Table 1.

119

120 Ethics approval

121 The institutional review boards approved the study, and written informed consent was

122 obtained from all participants.

123

124 RESULTS

125 Patient characteristics

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126 Following 'Interim Guidance, Clinical Management of COVID-19' published by the WHO

127 on 27 May 2020, one of the 12 patients was categorized as asymptomatic, one patient as mild,

128 three patients as severe; and the remaining seven patients as critical infection. Eleven patients

129 were >50 years old, and the remaining patient (K-Px-11, with a mild infection) was 40 years old.

130 Five of the seven patients in critical-infection group were male, and three of these five patients

131 have died as of this writing. The demographic and clinical data of patients are summarized in

132 Table 2.

133 We define 'COVID-19 critical patients' as those who suffer from acute respiratory distress

134 syndrome (ARDS) and require mechanical ventilation support. The duration of mechanical

135 ventilation support for 7 patients with critical infection in this study ranged from 10 to 50 days,

136 with shortest duration at 10 days in patient K-Px-6 who died after 16 days of hospitalization. In

137 general, the durations of hospitalization in patients with a critical infection were longer compared

138 to those of the other severity groups. There were two critically infected patients with relatively

139 short durations of hospitalization (16 days for K-Px-6 and 17 days for K-Px-7), but these

140 durations were short because the patients died at day 16 and day 17 after admission.

141 Among seven patients with critical disease, five had identified comorbidities related to

142 COVID-19 clinical severity; another had a medical history unrelated to COVID-19 severity

143 (osteoporosis), and the remaining patient did not have any background disease. All three patients

144 who died were males >70 years old who had significant comorbidities related to COVID-19

145 disease severity. Of the three patients with severe disease, two had hypertension as a comorbidity

146 related to COVID-19 severity. The mildly infected patient did not have any background disease.

147 Of note, one asymptomatic patient had diabetes mellitus as a comorbidity.

148

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149 The neutralizing antibody titer's trend in COVID-19 patients with different severities

150 Neutralizing antibody against virus is important for virus elimination. Therefore, to

151 examine neutralizing antibody production for SARS-CoV-2, we serially collected blood samples

152 from COVID-19 patients and determined the trend of neutralizing antibody titer against SARS-

153 CoV-2 in serum of each patient.

154 A total of 45 blood samples from 12 patients were subjected to the measurement of

155 neutralizing antibody activity. As shown in Figure 1, neutralizing antibody titer against SARS-

156 CoV-2 was detected in all 12 COVID-19 patients regardless of their disease severity, whereas

157 neutralizing antibody was not detected at all in healthy controls. The neutralizing antibodies in

158 10 patients with severe or critical disease showed relatively high titers compared to the

159 asymptomatic patient (K-Px-10), although patient with mild symptoms (K-Px-11) also showed a

160 high titer measured at day 42 after onset; this patient was a 40-year-old male who complained of

161 fever for 1 day before being confirmed as having COVID-19 by PCR. Although his case never

162 progressed to a more severe condition, low grade fever continued for a prolonged period. In

163 agreement with this patient's clinical course, PCRs were repeatedly performed from 19 to 34

164 days post-onset, and they showed positive and negative results alternately.

165 Our analysis of the induction of neutralizing antibody during the first days after disease

166 onset in patients K-Px-6, -7, -8 and -9 revealed that neutralizing antibody started to be induced

167 around day 4-10 post-onset, and titer then gradually increased, indicating that seroconversion had

168 occurred around these days. To elucidate the effect of neutralizing antibody production on the

169 persistence of virus replication, we investigated the time point(s) at which neutralizing antibody

170 started to be detected and virus genome became undetected. In all of the severe/critical patients

171 (excluding the two who died in which a follow-up PCR was not performed), neutralizing

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172 antibody had already been induced at the time that virus genome became undetected by PCR. A

173 different pattern was observed in patient K-Px-8, in whom viral genome could still be detected at

174 day 56 post-onset even though neutralizing antibody had already been induced. Of note, K-Px-8

175 is the only critical patient in this study who received corticosteroid pulse therapy from the

176 beginning of the disease course.

177 We thus observed that all the individuals infected with SARS-CoV-2 developed

178 neutralizing antibody, including the asymptomatic patient (although the titer was low).

179

180 The trend of cytokine/chemokine expression in patients with different COVID-19 severities

181 Cytokine release has been shown to be related to COVID-19 severity [8, 9]. We therefore

182 measured the levels of 48 different cytokines, chemokines, and growth factors (Table 1) in sera

183 of COVID-19 patients with different severities, at various time points. Figure 2 provides the

184 levels of the measured cytokines, chemokines, and growth factors in patients' sera, expressed in

185 picograms per milliliter of serum. In order to more easily analyze the trend of the individual

186 cytokine expressions in the different disease-severity groups, we have presented the measured

187 value of each cytokine or chemokine in a graded color scale (heat map). The average measured

188 values for each cytokine or chemokine in two healthy controls were also calculated, and the

189 values higher than those control values are scaled red-colored according to the amount.

190 Six of the seven critical patients required mechanical ventilation support after several days

191 of hospitalization, suggesting that disease progressivity continued during treatment. The day of

192 ventilator introduction may thus reflect the time point at which the disease clinically progresses

193 to a more severe condition, and the day of ventilator removal is likely to reflect the time point at

194 which patient shows sufficient clinical improvement in respiratory function. We thus analyzed

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195 the kinetics of cytokine/chemokine release in relation to the patients' clinical presentations. As

196 shown in patient K-Px-2, the levels of several cytokines and chemokines were higher during his

197 ventilator usage period (days 8–33 post-onset)—especially in the earlier days—compared

198 to levels beyond this period. A similar trend was observed in other critical COVID-19 patients

199 who recovered (K-Px4, K-Px-5, and K-Px-8), with the exception of patient K-Px-4, whose

200 cytokine and chemokine levels were constantly high for an extended period of time including

201 after recovery period.

202 For three critical COVID-19 patients who died (K-Px-3, K-Px-6, and K-Px-7), the trend of

203 cytokine/chemokine release was unique to each patient. Patient K-Px-3 showed consistently high

204 levels of most cytokines and chemokines from 1 week after the start of ventilator use until death

205 (day 18 until day 50 post-onset) with the following exceptions, which showed a reducing trend

206 toward death: IL-1ra, IL-5, IL-17, MCP-1, VEGF, IL-12p40, IL-16, HGF, IFN-α2, MCP-3, IL-6,

207 IL-18, and TNF-α.

208 A different pattern was observed in patient K-Px-6, with a clearly increased level of most

209 cytokines and chemokines in the days before death, emphasizing that this 'cytokine storm' was

210 the possible cause of death. Interestingly, patient K-Px-7, who had more comorbidities related to

211 COVID-19 severity compared to the other patients, did not show an increasing trend of cytokines

212 or chemokines toward death.

213

214 DISCUSSION

215 We analyzed the trend of neutralizing antibody production against SARS-CoV-2 and

216 cytokine/chemokine levels in a small series of COVID-19 patients with different severities. We

217 observed a clear neutralizing antibody response against SARS-CoV-2 in all 12 patients, whereas

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218 healthy controls did not show any neutralizing response at all, suggesting a highly specific nature

219 of neutralizing antibody against SARS-CoV-2. Critically and severely infected patients showed

220 higher neutralizing activity for SARS-CoV-2 compared to asymptomatic patient. This result is in

221 agreement with two reports that SARS-CoV-2-specific neutralizing antibody titer was higher in

222 patients with severe infections compared to those with mild infections [6, 7]. In the present

223 asymptomatic patient, a few increases in neutralizing antibody titer were seen, which may

224 indicate that virus replication was not very high or that it can replicate but only at local sites

225 without producing any symptoms. These results indicate that virus replication in severe cases

226 occurs in the lungs or systemically, and that in asymptomatic cases the replication is not very

227 high.

228 Despite being categorized as having a critical COVID-19 infection, patient K-Px-8

229 showed a relatively low neutralizing antibody titer against SARS-CoV-2 on multiple

230 measurements at different time points. This may be related to the use of methylprednisolone

231 pulse therapy from a relatively early phase of the disease (day 6 post-onset), at which time

232 neutralizing antibody may not yet be induced.

233 Although patient K-Px-11 was considered to have a mild infection, he continuously

234 presented with prolonged low grade fever, and serial viral genome detections showed positive

235 and negative results alternately, suggesting a continued replication of the virus for a relatively

236 long period. Therefore, the relatively high titer of neutralizing antibody detected in this patient is

237 not a surprising result.

238 By analyzing the trend of neutralizing antibody titer in these severe and critical COVID-

239 19 patients, we observed that neutralizing antibody against SARS-CoV-2 seems to be induced

240 around 4-10 days after disease onset. This observation is similar to that of a previous report of a

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241 young man with a mild SARS-CoV-2 infection in whom neutralizing antibody titer rapidly

242 increased from day 4 until day 20 [18]. Additionally, another report showed neutralizing

243 antibody against SARS-CoV-2 could be detected from day 10-15 after onset [19]. .

244 Not surprisingly, we also observed that neutralizing antibody was always induced before

245 SARS-CoV-2 genome testing showed a negative result, suggesting efficient viral clearance by a

246 successful induction of neutralizing antibodies. In addition, decreasing titer of neutralizing

247 antibody detected on day 33 in patient K-Px-5 was accompanied by clinical improvement, as

248 evident by ventilator weaning on day 32.

249 Regarding the assay of cytokine/chemokine release, our data showed clearly higher

250 amounts of most cytokines and chemokines in critical COVID-19 patients compared to patients

251 with milder symptoms (Fig. 2) at all time points. This observation is in agreement with reports

252 that cytokine storm is associated with COVID-19 severity [20].

253 IL-6, which plays a central role in the cytokine storm [9], signals through two main

254 pathways (cis- and trans-signaling pathways) which contribute to CRS [8, 21]. Consistent with

255 earlier reports, our present analyses demonstrated that serum IL-6 level was clearly increased in

256 all 10 of the severe/critical COVID-19 patients, but not in asymptomatic and mild patients.

257 However, our results indicated that determinant of disease severity was not only the induction of

258 IL-6, but also the induction of the other cytokines and/or chemokines.

259 Although most of the cytokines and chemokines tested herein were high in critical

260 COVID-19 patients compared to those with milder symptoms, some cytokines and/or

261 chemokines were detected at higher levels in some of the patients with milder symptoms. Among

262 these cytokines/chemokines were PDGF-bb, RANTES, IFN-α2, MIF, SCGF-β, and SDF-1α.

263 This result is inconsistent with a report that PDGF-BB, RANTES, and IL-9 were consistently

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264 low in the group of patients with severe and fatal infections compared to the mild-infection

265 group [22]. Our data showed that RANTES and PDGF-BB were not always higher in the non-

266 severe patients compared to the severe patients, whereas IL-9 was always detected at values that

267 were comparable to those of the healthy controls in all patients regardless of disease severity,

268 except for patient K-Px-4 who consistently showed relatively high levels of most of the

269 cytokines and chemokines over an extended period of time, including the recovery phase.

270 Based on the results of our analyses of the courses of COVID-19 patients' clinical

271 conditions, neutralizing antibody titers and cytokine/chemokine levels, we suggest the following

272 concept. In severe COVID-19 patients, SARS-CoV-2 replicates abundantly, especially in the

273 lung and/or systemically. The higher positivity rate of SARS-CoV-2 detection in sputum

274 compared to nasopharyngeal or throat swabs may be explained by the more efficient replication

275 of this virus in the lung [23-26]. Innate immunity is then induced in response to viral replication,

276 which is then followed by the induction of acquired immunity as shown by the detection of

277 specific antibody and T lymphocytes recognizing SARS-CoV-2. The induction of innate

278 immunity is reflected by the increased levels of cytokines and chemokines in serum, which are

279 responsible for the cytokine storm. Both virus replication and cytokine storm contribute to the

280 lung damage that occurs in patients with severe disease.

281 The induction of acquired immunity may then efficiently clear the virus, which is

282 evidenced by our observation that at the time the neutralizing antibody reached its higher level,

283 viral genomes turned undetected. However, by the time acquired immunity is induced, the

284 disease may still have been progressing continuously and lung damage continues as the cytokine

285 storm has not been controlled.

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286 Based on this understanding, we suggest that convalescent plasma therapy from COVID-

287 19 recovered patients to severely infected patients, human or humanized antibody therapy, or

288 effective antiviral agent (when available) may provide better outcomes when administered earlier

289 in the disease process before severe cytokine production begins. Once viral infection is

290 eliminated in severe cases, steroid therapy may be added to suppress the cytokine storm.

291 We used authentic SARS-CoV-2 to measure neutralizing activity in this study. Recent

292 studies generally used a pseudotyped virus, for the neutralizing assay[11, 12]. However there

293 were inconsistent results in neutralizing assay between pseudotyped and authentic viruses [13-

294 15], which may be caused by different presentation of S protein resulting from different

295 environmental condition [13]. Therefore, it seems that the authentic virus should be used to

296 examine the neutralizing activity against SARS-CoV-2.

297 The neutralizing antibody titer for SARS-CoV-2 was recently shown to decrease gradually

298 [27]. Our present findings demonstrated that in patients with more severe infections, neutralizing

299 titer is relatively high, and therefore patients' antibodies may be retained for a long period. It will

300 be informative to continue measuring these patients' neutralizing antibody titers and to examine

301 whether the antibodies can protect patients from a second infection of SARS-CoV-2.

302

303 Funding/Support

304 This work was partly supported by budget of Hyogo Prefectural Government.

305

306 Acknowledgement

307 We thank Kazuro Sugimura MD, PhD (Executive Vice President, Kobe University) for his full

308 support to promote this study. We also thank Yukiya Kurahashi, Zhenxiao Ren, Anna Lystia

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309 Poetranto, Salma Aktar, Jing Rin Huang, and Silvia Sutandhio (Division of Clinical Virology,

310 Center for Infectious Diseases, Kobe University Graduate School of Medicine) for technical

311 support in this study. We express our sincere gratitude for cooperation and participation of staffs

312 of Hyogo Prefectural Kakogawa Medical Center. We thank Research Foundation for Microbial

313 Diseases of Osaka University (BIKEN), Osaka University for providing SARS-CoV-2 strain.

314

315 Author Contributions: L.T and T.N equally contributed to this work.

316 Concept and design: L.T, T.N, Y.M.

317 Acquisition, analysis, or interpretation of data: L.T, T.N, S.F, S.I, K.F, M.N, J.A, Y.N, Y.M

318 Drafting of the manuscript: L.T, Y.M

319 Critical revision of the manuscript for important intellectual content: L.T, T.N, Y.M

320 Administrative, technical, or material support: S.F, K.F, M.N, J.A

321 Supervision: Y.N, Y.M

322

323 Conflict of interest

324 The authors declare no conflicts of interest associated with this manuscript.

325

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391

392

393

18 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.

394 Table 395 Table 1. List of cytokines, chemokines and growth factor analyzed in this study

IL-1β Eotaxin IL-1α MIF

IL-1ra FGF basic IL-2Rα MIG

IL-2 G-CSF IL-3 β-NGF

IL-4 GM-CSF IL-12p40 SCF

IL-5 IFN-γ IL-16 SCGF-β

IL-7 IP-10 CTACK SDF-1α

IL-9 MCP-1(MCAF) GROα TNF-β

IL-10 MIP-1α HGF TRAIL

IL-12(p70) PDGF-bb IFN-α2 IL-6

IL-13 MIP-1β LIF IL-8

IL-15 RANTES MCP-3 IL-18

IL-17 VEGF M-CSF TNF-α

396 IL, interleukin; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; 397 GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IP, interferon 398 gamma-induced protein; MCP, monocyte chemotactic and activating factor; MIP, macrophage 399 inflammatory protein; PDGF, platelet-derived growth factor; RANTES, regulated on activation, 400 normal T-cell expressed and secreted; VEGF, vascular endothelial growth factor; CTAK, 401 cutaneous T-cell-attracting chemokine; GRO, growth-related oncogene; HGF, hepatocyte 402 growth factor; LIF, Leukemia inhibitory factor; MCP, monocyte chemotactic and activating 403 factor; M-CSF, macrophage colony stimulating factor; MIF, macrophage migration inhibitory 404 factor; MIG, monokine induced by interferon-gamma, NGF, nerve growth factor; SCF, stem 405 cell factor; SCGF, stem cell growth factor; SDF, stromal cell-derived factor; TNF, tumor 406 necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand

19 medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. 407 Table 2. Demographic and Clinical Characteristic of Patients Hospitalization Upper Persistent Ventilator Ventilator Code Sex, Severitya Medical historyb period Respiratory Pneumonia imaging introduction removal Treatmente Equipmentf Outcome Age (dpoc) symptomd abnormalities (dpoc) (dpoc)

K-Px-10 M, 59 Asymptomatic DM, glaucoma 20 - N.D. N.D. - - - - Alive doi:

K-Px-11 M, 40 Mild - 40 + ------Alive https://doi.org/10.1101/2020.08.05.20168682 K-Px-12 M, 71 Severe AR, BA, BPH 35 + + + - - C - Alive Cervical cancer, HL, K-Px-1 F, 59 Severe HT, SAS 23 + + + - - C, F - Alive K-Px-9 F, 69 Severe HL, HT 21 + + + - - C, F - Alive K-Px-2 M, 57 Critical - 56 + + + 8 33 F, L/R CHDF, HD Alive Dead K-Px-3 M, 74 Critical DM, glaucoma, HL 44 + + + 11 50 C, F, L/R CHDF (51 dpoc) K-Px-4 F, 63 Critical Osteoporosis 80 + + + 16 65 C, F, L/R ECMO Alive All rightsreserved.Noreuseallowedwithoutpermission.

K-Px-5 M, 65 Critical HT, hyperuricemia 93 + + - 12 32 C, F, L/R CHDF Alive

Arrhythmia, CI, Dead K-Px-6 M, 79 Critical dementia 16 + + + 6 15 C, F, L/R - (16 dpoc)

AAA, AP, arrhythmia, Dead K-Px-7 M, 78 Critical ASO, CHF, CKD, DM, 17 + + + 8 21 C, F - c GB, HT (22 dpo ) ;

F, L/R, this versionpostedAugust6,2020. K-Px-8 F, 79 Critical DM, IgA nephropathy 73 + + + 0 36 Steroid CRRT Alive (6-9 dpoc)

H1 F, 59 Healthy - - N.D. N.D. N.D. - - - - - H2 M, 55 Healthy - - N.D. N.D. N.D. - - - - - H3 M, 58 Healthy - - N.D. N.D. N.D. - - - - - H4 M, 62 Healthy - - N.D. N.D. N.D. - - - - - H5 F, 45 Healthy - - N.D. N.D. N.D. - - - - - H6 F, 40 Healthy - - N.D. N.D. N.D. - - - - - H7 M, 65 Healthy - - N.D. N.D. N.D. - - - - - 408 a. Severity criteria was as follows; Mild: upper respiratory symptoms without pneumonia ; Moderate: pneumonia requiring no supplemental oxygen; Severe: requirement for 409 supplemental oxygen; Critical; requirement for intensive care including mechanical ventilation 410 b. AR, allergic rhinitis; BA, bronchial asthma; BPH, benign prostate hyperplasia; HL, hyperlipidemia; HT, hypertension; SAS, sleep apnea syndrome; DM, diabetes mellitus; CI, 411 cerebral infarction; AAA, abdominal aortic aneurysm; AP, angina pectoris; ASO, arteriosclerosis obliterans; CHF, chronic heart failure; CKD, chronic kidney disease; GB, The copyrightholderforthispreprint 412 gallbladder stone 413 c. dpo stands for days post-onset of symptoms. 414 d. Upper respiratory symptom included fever, cough and sore throat. 415 e. C: ciclesonide, inhaled corticosteroid; F: favipiravir, broad-spectrum antiviral for influenza; L/R: lopinavir/ritonavir, oral antiviral for human-immunodeficiency virus; Steroid: 416 Steroid pulse with 0.5 g methylprednisolone per day during the indicated period. 417 f. CHDF, continuous hemodiafiltration; HD, hemodialysis; ECMO, extracorporeal membrane oxygenation; CRRT, continuous renal replacement therapy 418 N.D.: Not determined

20 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.

419 Figure Legends

420 Fig. 1. Neutralizing antibody titer in the COVID-19 patients' sera.

421 Neutralizing antibody titers against SARS-CoV-2 were analyzed. Each panel is for the indicated

422 patient and includes results for the sera obtained at single or several time point(s) from admission

423 to hospital until discharge. The bars on top of each panel indicate the severity of symptoms in

424 chronological order. Light Blue: asymptomatic. Orange: upper respiratory symptoms without

425 pneumonia. Pink: pneumonia requiring no supplemental oxygen. Red: requiring supplemental

426 oxygen. Black: requiring intensive care including mechanical ventilation. Dark Blue: Getting

427 better with residual symptoms. The results of the neutralization assay of the control sera from

428 seven healthy donors are also shown. Number 0 in the panel indicates no detection of

429 neutralizing activity. Δ below the bar indicates time point(s) for cytokines/chemokines

430 measurement showed at Fig.2.

431

432 Fig. 2. Cytokine and chemokine levels in the COVID-19 patients’ sera.

433 The concentrations of 48 cytokines and chemokines in sera were measured by the multiplex

434 suspension array system with the Bio-plex Pro™ human cytokine screening 48-plex panel (Bio-

435 Rad). The blank-corrected observed values were translated to the concentration with the standard

436 curve for each cytokine or chemokine and further corrected by the dilution rate. The average

437 values of duplicate wells are shown. Values less than the detection limit were treated as Zero and

438 colored gray. The average value of the two healthy controls was calculated for each cytokine or

439 chemokine, and values lower than the average are pale blue. Values higher than the average are

440 scaled-red according to the amount.

441

21 Asymptomatic Mild Severe K-Px-10 K-Px-11 K-Px-12 K-Px-1 K-Px-9 9 9 9 9 9 PCR8 + - - PCR8 + - + - + -+ -- PCR8 - + -- PCR8 + -- PCR8 + - - Δ Δ Δ Δ Δ Δ ) 7 7 7 7 7 6 6 6 6 6 log3 5 5 5 5 5 4 4 4 4 4 3 3 3 3 3 Neutralizing Neutralizing 2 2 2 2 2

antibody titer ( titer antibody 1 1 1 1 1 0 0 0 0 0 0 5 10 15 20 0 10 20 30 40 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 Days post-onset Days post-onset Days post-onset Days post-onset Days post-onset Critical K-Px-2 K-Px-3 K-Px-4 K-Px-5 9 9 9 9 8 8 PCR8 + - PCR8 + - PCR + -- PCR + - - - Δ Δ Δ Δ Δ Δ Δ ΔΔ Δ Δ Δ Δ Δ Δ Δ Δ ) 7 7 7 7 6 6 6 6 log3 5 5 5 5 4 4 4 4 3 3 3 3

medRxiv Neutralizing 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. 2 2 2 All rights reserved. No reuse allowed without permission. 2

antibody titer ( antibody titer 1 1 1 1 0 0 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 100 Days post-onset Days post-onset Days post-onset Days post-onset Critical K-Px-6 K-Px-7 K-Px-8 9 9 9 9 Healthy 8 8 8 Donors PCR + PCR + PCR + + -- 8 Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ ) 7 7 7 7 ) 6 6 6 6 log3

5 5 5 log3 5 4 4 4 4 3 3 3 3

Neutralizing Neutralizing 2 2 2 2 Neutralizing Neutralizing

antibody titer ( titer antibody 1 1 1 1

0 0 ( titer antibody 0 0 0 0 0 0 0 0 0 0 0 ↑ ↑ 0 5 10 15 0 5 10 15 20 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 Days post-onset Days post-onset Days post-onset Donor 48 Cytokines/Chemokines (pg/ml) Eotaxi FGF GM- PDGF- RANTE IL- SCGF- ID Dpo IL-1β IL-1ra IL-2 IL-4 IL-5 IL-7 IL-9 IL-10 IL-12 IL-13 IL-15 IL-17 G-CSF IFN-γ IP-10 MCP-1 MIP-1α MIP-1β VEGF IL-1α IL-2Rα IL-3 IL-16 CTACK GROα HGF IFN-α2 LIF MCP-3 M-CSF MIF MIG β-NGF SCF SDF-1α TNF-β TRAIL IL-6 IL-8 IL-18 TNF-α n basic CSF bb S 12p40 β

Healthy H1 - 1.36 0 0 0.89 0 0 330.13 0 0 0.96 0 2.39 73.65 20.55 220.01 0 11.66 482.46 25.71 9.49 1137.74 239.98 9073.43 0 5.02 29.01 0 0 27.75 744.2 627.97 511.96 0 24.04 0.45 6.47 631.14 198.07 0 60.73 179118.7 975.16 399.19 107.16 0 54.71 15.35 57.91 control H2 - 1.17 0 0 1 0 2.09 381.75 0 0 1.11 0 4 80.57 32.86 178.55 0 20.4 478.54 22.27 6.62 2628.22 270.56 9025.73 0 15.49 57.01 0 6.13 44.11 475.07 617.31 1045.38 0 36.76 0 36.75 730.5 151.78 0 109.21 381438.2 991.25 440.34 141.79 0 42.02 83.33 72.7

Asymptomatic K-Px-10 25 1.27 0 0 1 0 5.95 317.26 0 1.92 0.81 0 2.71 79.24 39.93 48.31 0.85 11.02 255.72 33.61 1.47 3539.96 220.28 8598.85 0 66.67 35.99 0 6.13 109.28 938.3 642.47 577.94 0 27.6 0 16.93 367.02 173.96 0 72.75 216652.6 903.36 412.76 132.96 0 5.05 30.52 60.58

Mild K-Px-11 48 3.24 1877.57 1.38 1.44 5.43 10.79 342.25 0 6.13 4.24 285.97 7.07 118.61 38.38 467.8 2.14 26.29 797.79 81.77 16.33 6210.44 351.64 14796.76 314.27 28.03 62.87 0.5 143.64 598.94 1318.38 901.61 1109.67 7.09 80.18 1.72 20.44 2277.71 186.63 17.15 135.09 277780.3 1704.16 415.81 166.02 0 73.51 104.85 119.07

K-Px-12 34 1.36 290.3 0 1.44 0 22.79 362.2 0 1.82 1.41 0 4.97 94.9 46.17 106.39 0.5 18.18 1124.25 43.14 3.14 4139.63 237.44 10598.53 0 25.02 86.21 0.1 54.44 62.07 1073.54 791.07 949.61 3.21 48.74 1.13 16.87 285.33 531.73 0 108.03 277710.3 1209.08 446.15 138.31 4.52 17.22 42.28 83.34

Severe K-Px-1 23 2.25 1171.67 0 1.38 0 18.1 340.11 2.17 0.4 1.16 0 5.13 58.63 49.72 187.16 1.97 36.19 2049.88 197.95 6.02 1993.3 214.54 11480.01 0 37.12 157.39 0.26 43.13 85.43 922.2 731.66 1804.82 3.83 52.96 11.41 52.58 608.17 1476.24 2.97 167.94 384172.9 1077.98 389.72 175.88 8.75 42.42 73.61 74.77

K-Px-9 4 1.56 124.86 0 1.25 7.62 11.27 194.75 0 0 1.16 0 5.29 102.33 37.86 177.94 3.72 27.89 3142.87 94.66 5.44 3526.74 158.09 10777.86 0 11.96 148.73 0.4 12.92 53.47 826.11 410.37 1089.36 33.13 95.54 5.97 23.77 54.89 1106.73 9.04 81.79 141649.4 945.3 240.08 155.21 14.53 30.35 105.03 77.73 17 2.06 134.82 2.01 1.46 0 13.16 88.54 2.17 0.09 0.96 606.78 8.36 10.07 47.08 1499.96 2.63 15.95 131.66 105.49 117.07 6323.11 177.35 644.69 401.72 32.86 155.12 0.48 37.35 71.92 791.88 419.94 1408.01 11.71 86.96 1.26 58.19 91.95 397.89 16.45 164.7 281861.3 156.04 88.34 90.04 16.85 382.88 42.45 30.64

19 125.02 15170.33 18.58 4.51 0 13.16 372.33 8.81 5.93 2.44 560.09 18.19 65.36 123.42 2434.45 7.28 143.61 6898.61 74.64 251.78 3010.12 811.26 17168.5 498.31 115.06 401.93 2.71 802.02 420.12 1144.55 1080.09 27976.09 26.3 237.78 10.5 293.31 2411.65 8676.2 56.92 507.08 418004.1 1386.18 425.34 274.06 214.55 1280.32 1004.13 243.73 K-Px-2 32 5.65 8513.66 10.66 2.84 0 3.97 263.2 13.72 3.93 2.44 372.77 11.09 61.07 97.57 566.64 4.61 133.13 30881.25 70.76 27.24 770.94 246.28 3389.34 312.82 67.91 531.72 1.72 269.24 300.94 3886.4 618.13 2698.01 18.87 142.01 5.06 218.49 1261.06 36373.79 13.51 695.8 211132.1 1753.73 298.27 162.81 16.72 38.64 702.84 386.98 38 3.67 999.94 0 1.91 0 6.22 362.2 6.25 2.53 1.79 0 9.81 92.7 83.46 447.9 2.51 123.87 4957.31 69.97 17.42 2041.25 266.95 12669.17 0 45.06 458.35 0.58 143.64 168.38 1882.5 818.99 1366.14 10.83 105.85 2.41 215.93 1037.8 6448.45 11.27 763.98 281554.9 1500.75 427.55 209.57 30.08 38.84 558.54 160.94 54 5.53 983.12 0 1.91 0 3.37 368.07 3.95 1.32 1.6 0 8.52 110.05 73.15 603.7 1.71 49.52 3357.7 63.95 31.64 1666.16 296.32 12325.11 0 33.47 137.17 0.12 92.31 105.59 2228.55 809.99 947.36 8.07 88.88 1.6 133.89 1019.97 6618.16 9.18 373.96 293281.1 1324.45 434.54 209.34 2.84 41.42 461.61 156.51

18 5.74 13998.72 6.36 2.74 81.32 13.39 363.98 0.66 2.93 1.74 0 12.38 22.62 77.95 641.77 3.87 191.33 7234.32 781.01 26.25 6364.56 330.76 12510.38 459.06 66.67 865.43 0.59 315.67 419.17 1175.22 853.51 10007.33 12.13 94.59 62.18 174.17 1285.68 5078.01 13.23 321.73 390117.5 539.96 420.91 205.26 764.51 204.54 519.48 252.57 25 5.17 15415.67 7.18 2.45 0 31.64 356.86 0.66 3.43 1.79 0 10.61 104.83 87.86 495.58 3.33 148.37 2150.74 287.95 17.87 5941.99 264.52 13020.1 432.72 67.29 665.14 0.62 477.48 548.67 1276.78 1184.77 16561.03 13.6 103.99 25.71 186.4 651.1 1992.01 28.15 221.12 473970.6 1214.72 416.66 215.45 89.9 219.19 588.33 199.54 K-Px-3 36 3.79 3875.7 0 2.28 0 12.22 328.7 0 3.43 1.36 0 9.97 56.9 109.9 794.52 2.26 101.51 1978.58 43.87 38.92 3489.68 222.38 9847.95 0 48.74 433.22 0.24 486.27 263.14 1398.66 747.78 15131.98 10.39 86.96 3.14 116.08 342.98 3608.41 0 258.12 328844.7 947.22 401.9 187.53 59.17 242.26 171.58 136.47 48 4.27 3544.71 0 1.91 0 17.45 382.11 0 7.33 3.44 0 6.26 102.71 68.79 732.74 5.19 106.33 4302.23 59.29 34.87 5630.69 274.2 12900.26 0 35.9 556 0.17 73.65 261.58 2235.98 1215.57 2086.76 7.83 73.31 7.81 142.96 542.31 1862.85 10.72 226.47 326496.8 1387.21 428.91 205.72 60.62 243.09 159.87 137.65 50 8.01 3950.57 0 1.91 0 16.12 332.45 0.66 7.33 4.73 0 6.1 105.01 66.01 387.26 4.83 83.36 5176.52 81.39 17.32 5352.99 243.62 11938.72 0 37.12 413.53 0.23 81.7 211.05 1878.38 1201.86 1844.58 7.09 61.24 6.25 114.47 525.63 4468.93 9.74 233.2 290761.4 1452.51 399.36 204.81 59.35 130.58 165.85 151.8

19 7.3 10900.1 7.42 5.31 237.2 45.35 853.89 22.82 31.5 7.47 581.97 20.44 59.12 120.43 780.61 18.2 300.7 8355.45 830.3 28.2 6109.55 354.13 8588.12 555.44 142.46 499.36 1.58 231.46 507.2 757.9 1218.29 4538.1 25.11 274.41 110.96 144.21 1103.26 4084.55 48.2 191.73 416123.8 2281.86 939.93 327.39 112.3 169.93 408.99 265.53 K-Px-4 32 6.54 3102.01 6.71 4.49 175.32 25.26 771.73 18.65 30 6.37 268.35 22.22 89.41 155.62 461.55 14.89 224.77 2058.86 181.08 14.85 3191.58 324.22 11864.06 309.93 124.6 283.85 1.14 334.03 368.36 924.29 1017.28 8896.1 26.97 217.46 5.77 99.42 856.95 2110.79 31.55 254.31 332260.1 1917.59 835.72 235.98 35.75 64.42 214.29 161.23 72 6.12 1154.42 5.77 3.82 98.03 26.88 694.33 22.04 27.1 5.29 535.16 16.57 47.67 102.83 342.22 10 145.31 4158.8 124.05 9.68 3749.87 296.81 11780.25 365.42 122.69 234.43 1.07 185.68 123.83 948.15 1098.53 3183.19 23.2 191.79 4.42 68.04 923.08 4571.14 26.74 205.63 245897 1666.44 703.4 223.81 11.67 43.82 76.1 130.86

Critical 24 7.18 1710.74 5.77 3.29 5.43 5.12 296.14 4.46 5.73 1.84 0 23.67 141.05 110.4 669.72 2.94 277.09 8323.38 239.94 34.74 928.68 240.39 5119.84 0 89.17 1263.83 1.7 791.61 369.46 1101.39 417.57 30687.74 18.69 156.01 20.97 193.64 618.2 18152.7 0 498.45 248851.2 1130.9 348.32 202.09 103.22 243.21 616.2 149.15 30 4.36 1053.9 0 1.93 0 2.42 299.72 0.66 1.12 1.74 0 9.81 87.23 76.06 282.59 1.04 105 919.08 48.02 7.15 1211.32 187.3 8194.65 0 51.2 476.23 0.3 490.66 164.75 1000.18 241.92 17945.81 9.48 88.88 2.15 186.65 412.38 2280.16 0 462.96 255266.9 568.17 361.08 156.59 18.79 105.25 254.15 83.34 K-Px-5 37 2.72 653.74 0 1.46 0 2.75 296.49 1.16 1.32 1.65 0 7.87 97.46 67.76 250.58 1 45.82 411.51 77.72 6.46 1034.49 185.4 10107.25 0 32.26 169.35 0 315.67 105.59 929.13 570.17 9201.06 5.53 60.22 1.26 138.52 312.43 425.68 0 372.72 209871.4 614.98 345.64 138.08 38.51 166.21 133.23 91.62 62 2.34 721.73 0 1.67 0 14.99 352.23 3.69 2.12 1.79 0 8.19 168.39 45.25 331.76 2.22 105.5 2416.69 88.49 8.98 3688.31 229.4 11526.52 0 35.9 722.54 0.71 87.03 145.13 1287.05 646.44 2009.4 8.07 61.24 1.49 230.84 413.77 1757.75 12.67 306.34 442727.7 1391.33 439.49 181.14 12.93 39.63 646.2 119.07 101 1.72 1167.37 0 1.38 0 3.06 317.44 0 0 1.31 0 8.19 151.11 44.32 264.85 0.5 36.19 505.21 34.39 8.25 1329.72 192.82 7579.9 0 11.96 112.14 0 2.42 64.38 1140.25 391.83 800 0 35.64 0.45 48.92 61.45 659.13 0 181.79 248786.3 854.51 377.4 161.89 3.33 25.09 125.42 79.8

6 2.06 1868.12 0.73 1.62 0 14.99 304.56 16.57 1.12 1.88 191.84 6.58 105.62 47.08 263.66 1.76 42.77 16029.33 603.05 7.05 3420.1 198.53 7045.4 154.61 29.84 150.79 0.35 65.49 162.22 2024.87 800.9 1762.81 6.59 52.96 28.22 52.58 1614.23 2296.48 20.81 285.93 283918.5 1726.28 329.75 181.6 85.12 144.34 33.43 80.98 K-Px-6 8 3.18 4142.24 0 1.75 0 7.8 347.96 24.39 0.51 1.79 0 8.19 93.58 54.73 361.9 1.54 82.37 11827.56 381.75 13.47 1619.33 243.29 11547.07 0 34.68 372.14 1.47 65.49 196.27 1919.9 716.66 2303.15 8.07 57.13 19.71 46.36 1594.43 2087.72 8.07 245.13 311463.5 2086.99 394.79 175.65 212.64 96.08 77.56 107.26 15 7.01 20276.12 9.28 3.73 38.11 10.55 291.83 119.35 1.92 2.17 346.56 17.7 247.73 88.72 1622.53 5.19 479.42 30476.94 2038.64 115.38 2009.7 629.16 6025.5 293.72 77.89 2394.47 4.49 226.69 303.93 2407.12 991.09 6681.96 20.53 161.2 69.53 70.55 2687.23 15545.58 32.12 531.58 258359.4 2369.08 330.92 253.71 215.93 834.8 168.49 345.11

5 7.77 7091.25 3.25 1.75 1.62 14.08 363.62 19.69 2.33 1.5 153.42 8.03 105.54 61.31 305.5 3.72 67.83 12848.94 305.69 7.97 4736.79 246.2 12987.81 0 26.22 302.6 0.17 113.15 1086.18 1241.85 947.56 3862.86 9.71 76.27 15.67 71.53 3574.33 2768.21 26.74 77.94 342424.8 1555.54 404.78 185.25 97.74 47.64 220.21 105.49 K-Px-7 7 4.42 3360.8 4.46 1.62 0 19.83 315.66 14.76 2.73 1.5 0 6.91 73.92 56.72 244.62 3.1 57.92 8741.57 289.64 6.67 4829.2 209.86 11605.59 0 26.82 271.9 0.21 128.5 401.16 1451.15 809.35 4721.73 9.02 82.13 15.12 55.38 1609.27 2270.69 21.37 70.58 325408 1444.17 360.91 183.88 127.8 37.25 207.19 93.39 13 2.38 4504.78 0 1.62 0 8.31 292.73 2.42 0.3 1.36 0 8.19 56.3 58.66 324.77 1.97 74.19 3510.65 189.53 14.17 1737.01 212.6 6785.55 0 19.04 291.68 0.13 110.57 192.49 1526.05 465.03 3774.31 4.98 69.33 19.56 58.13 786.59 1611.16 7.93 85.98 229008.4 1812.17 323.07 126.89 224.31 52.89 272.51 104.61 18 2.81 186.3 2.76 1.67 0 6.22 249.81 3.31 1.12 1.5 317.91 5.46 22.92 47.53 292.59 1.54 80.08 917.76 276.15 9.09 3653.92 179.79 1282.16 0 11.96 742.4 0.14 138.61 266.44 1167.53 699.16 8798.1 4.98 67.32 14.38 181.32 723.19 833.75 14.35 123.4 343342.9 954.6 258.87 136.92 104.39 151.77 101.1 44.29

9 43.88 5721.88 4.82 2.69 0 24.03 331.2 8.56 2.02 4.55 268.35 11.42 260.06 81.04 1849.51 2.98 53.07 5137.95 284.81 152.84 4491.12 513.21 13807.96 334.11 46.28 201.08 0.53 163.57 1174.96 1279.81 1186.63 5412.75 10.39 112.32 22.96 49.89 2607.37 859.51 29 463.68 331909 1166.73 379.76 220.2 38.42 934.35 148.78 81.87 15 2.93 3118.24 0 1.87 0 6.22 277.45 21.65 0 3.26 0 7.55 106.27 72.16 552.58 0.75 111.66 3527.75 307.86 25.47 1397.23 209.9 7185.32 0 33.47 303.63 0.9 250.43 154.63 1867.83 233.81 6363.59 7.34 72.32 22.85 99.48 207.04 3523.7 0 740.94 280519.6 920.63 303.09 169.01 33.56 82.47 123.03 109.33 K-Px-8 22 10.04 5375.45 0.33 2.59 0 6.22 355.79 13.46 0 3.22 0 12.71 184.96 95.96 776.16 2.22 132.62 7605.56 205.28 34.68 1598.61 273.15 10639.24 0 61.7 474.36 0.74 388.48 261.26 1446.1 1005.06 12103.4 12.98 109.55 8.63 139.83 795.52 15634.8 0 543.32 323213.7 1083.19 423.3 212.06 49.65 382.36 322.42 196.89 27 49.01 5786.08 3.85 3.44 0 5.67 349.02 3.69 0.51 2.99 0 17.22 163.4 103.09 3286.17 3.06 103.83 5579.22 63.35 316.97 1915.87 800 9115.69 0 81.64 336.21 0.85 374.95 174.06 1302.31 855.97 10935.58 19.43 140.24 4.51 109.88 803.22 12424.31 6.96 560.52 247101.9 988.67 408.17 277.86 345.19 3264.25 302.22 224.29 69 2.69 235.8 0 1.67 0 4.84 367.18 1.53 6.43 4.42 0 43.1 200.13 63.87 558.77 1.14 42.93 1064.38 94.54 22.75 2236.19 238.57 10189.84 0 23.22 208.49 0.07 25.48 100.77 2022.59 822.82 1035.24 3.83 59.19 0.74 54.77 349.04 3410.52 12.95 550.74 237858.3 1384.81 414.96 145.96 8.25 141.35 58.89 67.68

Higher than the Low High Lower than the Less than the healthy control healthy control detection limit