key: cord-0972348-frkubjux authors: Marchi, S.; Simonetta, V.; Remarque, E.; Ruello, A.; Bombardieri, E.; Bollati, V.; Milani, G.; Manenti, A.; Lapini, G.; Rebuffat, A.; Montomoli, E.; Trombetta, C. title: Characterization of antibody response in asymptomatic and symptomatic SARS-CoV-2 infection date: 2021-03-31 journal: nan DOI: 10.1101/2021.03.29.21254534 sha: 8d34ee25cf0201d6059bd2134f0a5117fa754528 doc_id: 972348 cord_uid: frkubjux SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To elucidate the mechanism of infection, protection, or rapid evolution until fatal outcome of the disease we performed a study in hospitalized COVID-19 patients to investigate the time course of the antibody response in relation to the outcome. In comparison we investigated the time course of the antibody response in SARS-CoV-2 asymptomatic subjects. Study results show that patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies). The peak is reached by 3 weeks from hospital admission followed by a sharp decrease. No difference was observed in any parameter of the antibody classes, including neutralizing antibodies, between subjects who recovered or with fatal outcome. Only few asymptomatic subjects developed antibodies at detectable levels. with underlying comorbidities, particularly in the elderly, such as diabetes, hypertension, chronic 62 respiratory disease or cardiovascular disease [9, 12] . After almost one year after the first cases were 63 reported in Wuhan, as of 13 December 2020, there have been over 70 million cases and over 1.5 64 million deaths reported to WHO of confirmed with Europe being one of the most 65 affected areas, second only to the Americas. COVID-19 pandemic is causing high morbidity and 66 mortality burden worldwide, an unprecedented strain on health care systems, with social and 67 economic disruption as the only effective prevention is social distancing leading entire countries in 68 quarantine for weeks or months with dramatic impact on day-to-day human, social and economic life 69 [14] . 70 Italy has been one of the earliest and most affected countries by . Although the first SARS- CoV-2 case identified in Codogno at the end of February 2020 is considered the Italian index case, 72 some evidence has later arisen that the virus had circulated in Italy and Europe since autumn 2019 73 [15] [16] [17] [18] . Italy suffered the first wave from February until June 2020 when the whole country was 74 under strict lockdown. During this period the most affected areas were located in Northern and to a 75 less extent in Central Italy, while the Southern part of the country was relatively unaffected [19, 20] . 76 During the summer period until the end of September 2020 COVID-19 remained endemic, with a 77 second epidemic wave starting in October 2020 that led to a second national lockdown in November 78 2020. As of the 13 th of December 2020, more than 1.8 million confirmed cases and more than 64.000 79 deaths due to SARS-CoV-2 were reported to ISS (Istituto Superiore di Sanità) , Rome [21] . The mean 80 age of fatalities in COVID-19 infected people was 80 years, 42,4% were women and more than 90% 81 had one or more co-morbidity as ischemic heart disease, diabetes, active cancer, atrial fibrillation, 82 dementia, and a history of stroke [19] . 83 The emerging and rapid diffusion of COVID-19 has risen the calls for more targeted research in the 84 field [22] helping to elucidate the mechanism of infection, protection, or rapid evolution until fatal 85 outcome of the disease. We present here a study performed in hospitalized infected patients to investigate the time course of the antibody response in relation to the outcome, and as 87 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 explorative comparison, we also investigated the time course of the antibody response in SARS-CoV-88 2 asymptomatic subjects. 91 This was a retrospective study on COVID-19 patients and SARS-CoV-2 asymptomatic subjects 92 during the first epidemic wave occurred in Italy between March and May 2020. A total of 42 COVID-19 patients, hospitalized at Humanitas Gavazzeni (Bergamo, Italy), were 94 retrospectively selected for this study, of whom 35 (22 males and 13 females) recovered and 7 (3 95 males and 4 females) had a fatal outcome. All subjects were admitted to hospital with a diagnosis of 96 interstitial pneumonia confirmed by chest radiograph or a CAT (computerized axial tomography) and 97 had rhino-pharyngeal swab positive to SARS-CoV-2 (Real-Time PCR Thermo Fisher Scientific). Six 98 patients required care in the intensive care unit (ICU), the others were hospitalized in the general 99 medicine unit. Out of 7 deceased patients, 3 were hospitalized in ICU and 4 in the general medicine 100 unit. Serum samples were collected at different time points from March to April 2020 for 102 diagnostic/therapeutic purposes. We selected patients who had available at least 5 blood samples 103 during the period of hospital admission (baseline, day 2, day 6, day 12-14, day 18-20, day 27-30). Demographic and clinical variables reported in this study were those collected at hospital admission. For the purpose of this study patients were categorized according to the outcome: recovered or 106 deceased. During the first phase of the COVID-19 epidemic, little was known about this novel CoV and there 108 was no standard therapy, so the management changed over the time. The Italian Society of Infectious 109 and Tropical Diseases recommended as therapy hydroxychloroquine, antiviral agents, steroids, low is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Serum samples from 25 asymptomatic subjects who presented a positive nasal swab for SARS-CoV-114 2 were collected as part of the UNICORN project and were analysed in the present study [23] . All serum samples were tested by commercial ELISA for the detection of IgA, IgG, and IgM against 123 the S1 of SARS-CoV-2 (Aeskulisa ® SARS-CoV-2 S1 IgA, IgM, IgG, Aesku.Diagnostics, 124 Wendelsheim, Germany) and for the detection of IgG against the nucleoprotein (NP) of SARS-CoV-125 2 (Aeskulisa ® SARS-CoV-2 NP IgG, Aesku.Diagnostics, Wendelsheim, Germany). According to the manufacturer's instructions, quantitative analysis was performed by use of a 4- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint were used. Seroconversion rates were compared using Fisher's exact test. Antibody levels were 147 statistically evaluated using t-tests. Statistical significance was set at p<0.05, two tailed. and 2.0 (SD 1.41), respectively, and comorbidities were indicated in 1.88 (SD 1.36) and 3.5 (SD 3.54) 157 of recovered and deceased patients, respectively. No differences were found between recovered and 158 deceased patients when compared for symptoms at admission (fever, cough, diarrhea, dyspnea) or 159 presence/absence of comorbidities and/or preexisting conditions. The other demographic, clinical, 160 and blood chemistry variables collected at baseline were similar between the two groups, with 161 exception of ALT that showed to be statistically significantly higher (p-value 0.021) in subjects who 162 recovered (Table 1a) . At hospital admission, 15 subjects (35.7%) were negative for S1 IgM, 11 (26.2%) for S1 IgA, 13 164 (30.9%) for S1 IgG, 15 (35.7%) for NP IgG, and 10 (23.8%) for neutralizing antibodies. Five subjects 165 (11.9%) were negative to any antibody assay at the time of admission; of these, 2 died and 3 166 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101/2021.03.29.21254534 doi: medRxiv preprint recovered. Two days after admission, 6 subjects (14.3%) were still negative for S1 IgM, 7 (16.7%) 167 for S1 IgA, 4 (9.5%) for S1 IgG, 3 (7.1%) for NP IgG, and 5 (11.9%) for neutralizing antibodies. Two subjects (4.8%) were still negative to any antibody assay; of these, 1 died and 1 recovered. At 6 169 days of sample collection, all subjects except one (97.6%) were positive to all assays (Figures 1-5 ). The exception was represented by a 40-year-old male subject, showing neither S1 IgM nor S1 IgA 171 positivity in any sample, NP IgG with a borderline result at admission and at day 2, and negative at 172 day 6, and neutralizing antibody titers less or equal than 40. This subject presented fever and dyspnea 173 at admission with no comorbidities or preexisting conditions and recovered in 12 days. Two subjects, one recovered and one deceased both within 6 days after admission, were both negative 175 to NP IgG at admission and at day 2. Two subjects, both recovered, were positive only for neutralizing 176 antibodies at admission, with titers less or equal to 40. Both showed positivity to all antibodies tested 177 at day 2 and later (Figures 1-5). Neutralizing antibodies were found in all patients, with a range from 10 to 5120. Antibody titers for patients are presented in table 2. S1 and NP antibodies started increasing at day 2 180 and again at day 6. A decrease for all antibodies was observed in recovered patients at day 27-30. S1 Considering seroconversion rates in comparison to baseline (Table 3) , IgM seroconversion rate 190 appeared higher in the deceased (p-value 0.043). No statistically significant difference in antibody titers at baseline and by peak antibody level (all 5 192 assays combined) was found between those survivors and deceased by using the Cox proportional is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101/2021.03.29.21254534 doi: medRxiv preprint hazard model. A good correlation was found among all assays ( Figure 6) . Overall, the level of S1 194 specific response was well correlated among antibody types (r=0.781 and r=0.794, S1 IgG correlating 195 with S1 IgA and S1 IgM, respectively; r=0.760 S1 IgA correlating with S1 IgM). S1 IgG response 196 was highly correlated with NP IgG (r=0.834). Neutralizing antibodies well correlated with all ELISA 197 antibodies tested (r=0.722 with S1 IgA, r=0.798 with S1 IgM, r=0.739 with S1 IgG, and r=0.730 with 198 NP IgG). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint (Table 1b) . Twenty-one subjects had the swab positive to SARS-CoV-2 in March 2020 and 216 a blood draw, of whom 19 subjects had a blood draw in May and 14 another blood draw in September. Four subjects had the swab positive to SARS-CoV-2 in May and a blood draw, of whom 1 had a 218 second blood draw in September. Out of 25 asymptomatic subjects, 16 (64.0%) were negative to any antibody at any time point. Nine 220 subjects (36.0%) had at least one detectable antibody type at least at one of the time points. At the 221 first time point 6 subjects (24.0%) had positive S1 IgG, of whom 2 also had S1 IgA and NP IgG, 1 222 also S1 IgM, S1 IgA and NP IgG and 1 had S1 IgA, NP IgG and positive neutralizing antibodies. Three subjects negative at any antibody at the first time points had antibodies at one of the subsequent 224 time points. One of these subjects was positive at any antibody assay at the second time point 225 including neutralizing antibodies. The other 2 subjects had S1 IgA, S1 IgG and NP IgG at the third 226 time point. One subject was positive to all ELISA antibodies (S1 IgM, S1 IgA, S1 IgG, and NP IgG) available. Both subjects were also positive to S1 IgA, S1 IgG, and NP IgG; only the first one was also 231 positive to S1 IgM. Asymptomatic subjects with positive antibody levels in any of the assays had titers well below the 233 level found in symptomatic subjects as shown in figures 1-5 (table 4) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101 /2021 In this study we primarily evaluated the time course of the antibody response to different antigens of 259 SARS-CoV-2 (IgG, IgM, and IgA against S1, IgG against NP, and neutralizing antibodies) in 260 COVID-19 patients admitted to hospital for pneumonia during the first epidemic wave in March and 261 April 2020 in Italy. No significant difference in titers was observed in any of the S1 antibody class at 262 any time point between patients who survived and who did not survive. However, in deceased 263 patients, a sharper increase of S1 antibodies has been observed suggesting a potential risk factor. Notably, IgM seroconversion rate appeared somewhat higher in the deceased, which might indicate 265 that the deceased were admitted early after infection. In other similar studies early antibody response 266 to S1 IgA or IgM or difference in the magnitude of the immune response to SARS-CoV-2 infection 267 was a predictor of disease severity or progression or outcome [25] [26] [27] [28] . IgG antibody titers against NP other factors could be implicated in the specific immune response against SARS-CoV-2 that need to 275 be further investigated, as other elements of cellular-mediated immunity may play a role in protection. The kinetics of the antibody response showed an increase starting from day 2 and reaching the peak 277 between days 6 and 18-20. At 27-30 day, a decline in titers was observed for any of the antibody 278 classes. In other studies, the antibody kinetic in COVID-19 patients showed the antibody peak 279 reaching up to the 4 th and 5 th week from disease onset followed by antibody decay starting at the 6 th 280 week [28, 29] . This observation differs from our findings most likely as our study period of patients 281 started at hospital admission when the severity of the disease was already in an advanced stage. In this study, a high correlation between S1 and NP protein-based ELISAs and the neutralization 283 assay was observed in COVID-19 patients. The reason for that is likely due to the fact that the S1 284 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101/2021.03.29.21254534 doi: medRxiv preprint protein represents the immunodominant antigen of SARS-CoV-2 virus and functional neutralizing 285 antibodies are mainly targeting the Receptor-Binding Domain, a subunit of that protein [30] . In this study, only few asymptomatic subjects had detectable titers with almost two-thirds negative at 287 any time point. These findings support the observation that the use of serology testing for population 288 surveys might account of false negative results as asymptomatic or paucisymptomatic subjects might 289 have antibody concentration below the level of detection [31] . Only few asymptomatic subjects had 290 detectable titers to S1 IgG or more antibodies but with a low level of titers as compared to COVID-291 19 patients included in this study in accordance with another study [32] . Notably, in contrast to 292 COVID-19 patients, the majority of asymptomatic subjects do not have circulating neutralizing 293 antibodies that are considered a surrogate of protection against COVID-19, thus vaccination is highly 294 recommended. Although further evidence should be provided to establish a correlate of protection, 295 based on these data it can be speculate that evidence of neutralizing antibodies might be used as 296 markers of protective immunity. Asymptomatic subjects have a low viral load in the nasopharynges as assessed by RT-PCR and most 298 likely a lack or a defective viral replication and/or mucosal invasion that induces a weak or any 299 antibody response [33] . Although memory B and T cells may have been primed in SARS-CoV-2 300 swab positive subjects with undetectable antibodies in serum, with the ability to induce a rapid 301 immune response to re-exposure, the question of whether these subjects should be vaccinated is 302 critical now that effective vaccines are available against COVID-19. One limitation of this study is its retrospective nature and the collection of COVID-19 samples was 304 carried out only in a single center. Besides this, we limited the collection to those patients for whom 305 it was possible to construct an antibody response curve over a period of at least one month. This, of 306 course, represents a bias, however, the ratio between deceased and recovered patients (7 out of 42), 307 16.6% falls in the range from 5.7% to 30.4% as described in the literature [34, 35] . This value shows 308 high variability because it can be influenced both by the characteristics of the series studied and the 309 different treatments. The findings from this study do not allow us to predict the kinetics of the is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 antibody decay over time in patients who recovered from COVID-19, in particular, who will be 311 susceptible to reinfection over time, since no follow-up samples after discharge were available. Overall, our data highlight that COVID-19 patients produce a simultaneous antibody response to 313 SARS-CoV-2 with high correlation between different viral antigens (S1 and NP) and among antibody 314 classes (IgA, IgG, and IgM and neutralizing antibodies). The peak is reached by 3 weeks from hospital 315 admission followed by a sharp decrease. On the contrary, only few asymptomatic subjects developed 316 antibodies at detectable levels, though significantly lower compared to COVID-19 patients. Since 317 neutralizing antibodies were rarely produced, this finding raises the question about the protection of 318 these subjects against reinfection. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101/2021.03.29.21254534 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 World Health Organization. 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Titres are shown as log-2 transformed This study was funded by a research grant (Pfizer Tracking Number 60353289) . 332This funding source did not have any role in the design of this study and during its execution, analyses, 333interpretation of the data, or decision to submit results. 334This study was supported by the European Virus Archive goes Global (EVAg) project, which has received 335 funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 336No 653316. 337The UNICORN population was recruited thanks to the Funding Action "Ricerche Emergenza coronavirus", 338University of Milan, 2020. 339The authors thank Linda Benincasa for technical support. 340 341 342