key: cord-0824548-x60ebqm8 authors: Anichini, Gabriele; Gandolfo, Claudia; Terrosi, Chiara; Fabrizi, Simonetta; Miceli, Giovanni Battista; Gori Savellini, Gianni; Prathyumnan, Shibily; Franchi, Federico; Cusi, Maria Grazia title: Antibody response to SARS‐CoV‐2 in infected patients with different clinical outcome date: 2021-01-26 journal: J Med Virol DOI: 10.1002/jmv.26789 sha: bec0e9baa9459d95d2881fac6f4e642f534d4a4c doc_id: 824548 cord_uid: x60ebqm8 Data regarding antibody responses to severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) in patients infected with COVID‐19 are not yet available. In this study, we aimed to evaluate serum antibody responses in patients regardless of the outcome. We measured the circulating immunoglobulin G (IgG) antibody levels in 60 subjects with a certified history of SARS‐CoV‐2 infection by using immunoenzymatic, chemiluminescent, and Neutralization assays. Half patients had a severe infection, the other half were pauci‐symptomatic. We analyzed their antibody response to see the trend of the humoral response. Our results showed a significant difference in circulating IgG level among the two groups. The neutralizing antibody response against SARS‐CoV‐2 was significantly higher among those who had severe disease. Furthermore, ten subjects from each group were screened twice, and a declining antibody trend was observed in pauci‐symptomatic individuals. These findings provide evidence that humoral immunity against SARS‐CoV‐2 in pauci‐symptomatic people is weak and may not be long‐lasting. This may have implications for immunity strategy and prevention, since it is still not clear whether a time‐dependent decrease of both circulating and neutralizing antibodies to nonprotective levels could occur in a longer time span and whether potential vaccines are able to induce a herd immunity and a durable response. Data regarding antibody responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in patients infected with COVID- 19 are not yet available. In this study, we aimed to evaluate serum antibody responses in patients regardless of the outcome. We measured the circulating immunoglobulin G (IgG) antibody levels in 60 subjects with a certified history of SARS-CoV-2 infection by using immunoenzymatic, chemiluminescent, and Neutralization assays. Half patients had a severe infection, the other half were pauci-symptomatic. We analyzed their antibody response to see the trend of the humoral response. Our results showed a significant difference in circulating IgG level among the two groups. The neutralizing antibody response against SARS-CoV-2 was significantly higher among those who had severe disease. Furthermore, ten subjects from each group were screened twice, and a declining antibody trend was observed in pauci-symptomatic individuals. These findings provide evidence that humoral immunity against SARS-CoV-2 in pauci-symptomatic people is weak and may not be long-lasting. This may have implications for immunity strategy and prevention, since it is still not clear whether a time-dependent decrease of both circulating and neutralizing antibodies to nonprotective levels could occur in a longer time span and whether potential vaccines are able to induce a herd immunity and a durable response. "severe acute respiratory syndrome coronavirus-2" (SARS-CoV-2). As an emerging acute respiratory infectious disease, SARS CoV-2 primarily spreads through the respiratory tract, by droplets, respiratory secretions, and direct contact, 3 with a high human-tohuman transmissibility. Most adults or children with SARS-CoV-2 infection present mild flu-like symptoms; only a minority of patients have a severe outcome and rapidly develop acute respiratory distress syndrome, respiratory and multiple organ failure, bleeding and coagulation dysfunction, even death. 4 So far, the golden clinical diagnostic method of COVID-19 is nucleic acid detection in the nasopharyngeal swab or other lower respiratory tract samplings by real-time PCR, which can be further confirmed by next-generation sequencing. Apart from RT-PCR testing, serological testing is an additional emerging option in COVID-19 diagnostics 5 primarily as a proof of past infection but also to support the diagnosis of suspected COVID-19 patients. 6, 7 Serological assays for the evaluation of the humoral responses against Spike (S) and Nucleoprotein (N) in COVID-19 patients have been assessed, because of their high immunogenicity. Spike plays an important role in viral binding and entry into target cells, 8 while the Nucleoprotein in viral replication and assembly. 9 The kinetics of anti-N response has been described as similar to that of the anti-S, although N responses might appear earlier. 7 Anti-SARS-CoV-2 antibody titers seem to correlate with disease severity, likely reflecting higher viral replication rates and/or immune activation in patients with severe outcome. 10 In hospitalized patients, seroconversion is typically detected between 5 and 14 days postsymptoms onset, with a median time of 5-12 days for anti-S immunoglobulin M and 14 days for immunoglobulin G (IgG), and immunoglobulin A. 6, 7, 11, 11, 12 Neutralizing antibodies have been detected in symptomatic individuals 13, 14 and their potency seems to be associated with high levels of circulating antibodies. On the other hand, despite representing the majority of SARS-CoV-2 infections, asymptomatic infections are currently poorly documented 15 and whether this immunity is mediated by neutralizing antibodies remains an outstanding question . 16 Moreover, it is still unknown how long SARS-CoV-2 infected subjects could maintain long-term immunity and long-lasting protective antibodies, regardless of the outcome. In this study, we measured the circulating IgG antibody levels in 60 subjects with a certified history of SARS-CoV-2 infection, by using three different assays based on different methods. Subjects were equally divided into two groups: those hospitalized, who had a severe outcome, and those pauci-symptomatic. We analyzed their antibody response to see the trend of the humoral response in individuals with different disease outcomes. Moreover, 10 patients of each group were screened a second time to evaluate the persistence of anti-SARS-CoV-2 antibody 2 months after symptoms onset. The participants in this study were subjects with an assessed history of SARS-CoV-2 infection, between March and May 2020. Half of them were hospitalized in "Santa Maria alle Scotte" University Hospital, in Siena with a severe outcome. Instead, the other half consisted of pauci-symptomatic subjects reporting mild signs compatible with COVID-19 (fever, cough), who were placed in isolation at home. All infections were confirmed by RT-qPCR Test Subjects' sera were analyzed using two separate immunoassays. The Geometric mean titers (GMTs) of the neutralization assay were calculated. The differences between age, time of blood sample collection, circulating IgG levels, and neutralizing titers were evaluated and the statistical significances assessed with two-tailed χ 2 test. Results were considered statistically significant at p < .05. Spearman's rank correlation coefficient was used to assess correlations of log-transformed continuous variables between the groups. Results obtained by ELISA and CMIA, respectively, showed a significant difference in circulating IgG level among patients with a severe outcome and those with mild symptoms at T 0 (n = 30; 7.31 vs. 4.06; p = .0018 and 6.21 vs. 4.95; p = .048) (Table 1A) . Concerning those subjects who were screened twice (n = 10+10), no significant differences in IgG levels were found between the two samplings (T 0 , T 1 ) of both the groups, using both ELISA and CMIA (p > .05), although a lower IgG level was noticed among those with mild symptoms (Table 1B) . On the contrary, regarding the neutralizing activity, an evident GMT difference was found between the two groups at T 0 ( Figure 1 ); indeed, a higher titer was present in severe cases in comparison with those having mild disease (87.7 vs. 23.3; p = .0002). This difference was also confirmed for the patients tested twice (p = .046), although no significant difference in neutralizing antibody titer was found between the first and the second samples drawn 1 month apart from the same subjects, probably due to the limited number of samples (Table 1B) . It is worthy mentioning that a different trend of antibody response was observed in the H group, where the tendency of neutralizing antibodies was increasing over time, while it was decreasing in pauci-symptomatic individuals. We correlated the IgG titers obtained in the two serological assays, ELISA and CMIA, to the neutralizing antibody titers, to evaluate whether circulating IgG antibody levels could partly be associated to a neutralizing activity. As expected, we observed a moderate positive correlation between the neutralizing response and circulating IgG by using the whole virus proteins-based ELISA (r = .60) and a weak correlation using SARS CoV-2 N antigen-based CMIA (r = .44) (data not shown). In this study, we analyzed the titer of anti-SARS-CoV-2 IgG antibodies with three different serological assays in a cohort of subjects with a certified history of COVID-19, equally distributed with a severe outcome or mild symptoms. Despite the limited number of subjects, the most remarkable finding of this study was the significantly lower antibody titer in patients who experienced mild infection with respect to those affected by a severe respiratory syndrome. Both ELISA and CMIA, although based on different antigens, such as all virus proteins in the first assay and the nucleoprotein in the second one, showed an antibody response, which was significantly higher in patients with severe disease than in pauci-symptomatic subjects in the same time frame since symptoms onset. Previous studies on humoral response in SARS and MERS demonstrated that the humoral response could wane over time. 18, 19 We do not know how long this immunity could last in individuals affected by However, although the decay of total specific IgG was similar in both the groups, we noticed that the neutralizing antibodies, representing the protective response, only raised in severe cases 2 months after symptoms onset. On the contrary, the neutralizing response was very low in paucisymptomatic individuals (GMT: 29.2) with an evident decrease after 2 months (GMT: 21.52). These data raise concern that humoral immunity against SARS-CoV-2 may not be long-lasting in people with mild illness, threatening their protective status. Moreover, neutralizing antibody titers from all study subjects did not show a good correlation with the level of circulating IgG antibodies evaluated in ELISA or CMIA. In particular, only a modest correlation (r = .60) was found with ELISA values, while weak correlation (r = .44) was shown with CMIA. This can be easily explained on the basis of the antigen used in each test. Indeed, ELISA was based on all the viral proteins, including the Spike protein, responsible for cell binding to the receptor and containing the sequence recognized by neutralizing antibodies. CMIA was only using the immunogenic nucleoprotein, to which the humoral response is promptly mounted in the host, but it is not involved in the neutralizing activity. The observational time considered in this study was quite short, but the preliminary results indicated that a part of the population, particularly young people who presented a very mild disease, developed a weak humoral response, mainly characterized by a low neutralizing activity that could wane over time. For this reason, patients with high levels of circulating antibodies, especially those who had a severe outcome, could be more likely protected, while subjects with a favorable outcome, who showed low levels of neutralizing antibodies, may not maintain a long-lasting response and be susceptible to reinfection. Therefore, it could be important to keep monitoring both kind of subjects and see This finding may have implications for immunity strategy and prevention, since it is still not clear whether the immunity is longlasting and the potential vaccines, based on the spike antigen, are able to induce a durable response and herd immunity. Further studies are necessary to understand the role of cellular immune response and identify the correlates of protection for COVID-19. T A B L E 1 Serological analysis of the study population according to the clinical outcome in subjects screened once (Table 1A) and twice (Table 1B) Severe cases (H) Pauci-symptomatic (P) F I G U R E 1 Differences in neutralizing antibody titers between SARS-CoV-2 infected patients with mild or severe outcome. The whiskers represent the values from the 5th to the 95th percentiles; the GMTs are depicted by the horizontal lines in the boxes. Individual data points are shown. The p value of the GMT between the two groups is 0.0002. GMT, Geometric mean titer; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2 China novel coronavirus investigating and research team. 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