key: cord-341396-0tn06al2 authors: Ni, Ling; Ye, Fang; Cheng, Meng-Li; Feng, Yu; Deng, Yong-Qiang; Zhao, Hui; Wei, Peng; Ge, Jiwan; Gou, Mengting; Li, Xiaoli; Sun, Lin; Cao, Tianshu; Wang, Pengzhi; Zhou, Chao; Zhang, Rongrong; Liang, Peng; Guo, Han; Wang, Xinquan; Qin, Cheng-Feng; Chen, Fang; Dong, Chen title: Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals date: 2020-05-03 journal: Immunity DOI: 10.1016/j.immuni.2020.04.023 sha: doc_id: 341396 cord_uid: 0tn06al2 Summary The World Health Organization has declared SARS-CoV-2 virus outbreak a world-wide pandemic. However, there is very limited understanding on the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. Here, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and detected SARS-CoV-2-specific humoral and cellular immunity in 8 newly discharged patients. Follow-up analysis on another cohort of 6 patients 2 weeks post discharge also revealed high titers of IgG antibodies. In all 14 patients tested, 13 displayed serum neutralizing activities in a pseudotype entry assay. Notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific T cells. Our work provides a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in developing an effective vaccine to SARS-CoV-2 infection. *These authors contributed equally to this work. 1 2 #To whom correspondence should be addressed: Chen Dong, chendong@tsinghua.edu.cn; 3 or Fang Chen, anzhenchenfang@163.com; Cheng-Feng Qin, qincf@bmi.ac.cn. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 1 Introduction 1 At the end of 2019, patients with Coronavirus Disease 2019 were identified 2 in Wuhan, China (Wang et al., 2020) , infected by a novel coronavirus, now named as severe 3 acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The World Health Organization 4 (WHO) first declared this outbreak a public health emergency of international concern 5 (Phelan et al., 2020) and subsequently a world-wide pandemic (Di Pierro et al., 2020) . The genome sequence of SARS-CoV-2 bears 96% (Zhou et al., 2020) and 79.5% 7 identity to that of a bat coronavirus and SARS-CoV, respectively (Zhu et al., 2020) . Like 8 SARS-CoV and MERS-CoV, SARS-CoV-2 belongs to the beta genus Coronavirus in the 9 Corornaviridae family (Lu et al., 2020) . Clinically, several papers showed that most COVID-10 19 patients developed lymphopenia as well as pneumonia with higher plasma levels of pro-11 inflammatory cytokines in severe cases (Chan et al., 2020; Huang et al., 2020; Wu et al., 12 2020), suggesting that the host immune system is involved in the pathogenesis (Mahallawi 13 et al., 2018; Nicholls et al., 2003) . Patients infected by SARS-CoV or MERS-CoV were 14 previously reported to have antibody responses (Ko et al., 2017; Shi et al., 2004; Wang et al., (Thevarajan et al., 2020) . One COVID-19 patient in 1 Finland was shown to possess a low level of neutralizing antibody titer (Haveri et al., 2020) . However, virus-specific T lymphocytes and their relationships with neutralizing antibody 3 titers in COVID-19 patients remains uncharacterized. In this study, we collected blood from COVID-19 patients who have recently become 5 virus-free and therefore were discharged, and analyzed their SARS-CoV-2-specific antibody 6 and T cell responses. whereas the remaining 6 were 2 weeks post discharge (follow-up patients, patients #9-14). Only three travelled to Wuhan city within the past 3 months. In line with the previous reports 16 (Wang et al., 2016) , 2 patients (#5, 10) showed lymphopenia (normal range is 1.1-3.2X10e9 17 cells per L). Sera from three healthy donors (Wang et al., 2016) were obtained before the 18 SARS-CoV-2 outbreak (healthy donor #1-3). 3 additional healthy donors (#4-6) who had 19 been in close contacts with the patients were recruited in this study. Human AB serum 20 collected from healthy male AB donors in the US (GemCell, CA) was used as a negative 21 control. In order to detect anti-viral immune responses, we first constructed recombinant pET28-23 N-6XHis by linking 6 copies of His tag to the C-terminus of NP in the pET28-N vector 24 (Biomed, Cat. number: BM2640). Escherichia coli transformed with pET28-N-6xHis was 25 lysed and tested by Coomassie blue staining to confirm NP expression at 45.51 kDa. NP 26 was further purified by Ni-NTA affinity chromatography and gel filtration. The purity of NP 27 was approximately 90% ( Figure S1A ). The presence of NP was subsequently confirmed by 28 anti-Flag antibody ( Figure S1B ). The receptor-binding domain (RBD) of S protein (S-RBD) 1 and main protease (Lan et al., 2020) were produced by a Baculovirus insect expression 2 system and purified to a purity of 90% ( Figure S1A ). Using sera from patients and healthy donors, IgG and IgM against SARS-CoV-2 NP, 4 main protease and S-RBD antigens were analyzed. There was no significant antibody 5 response to main protease in sera from several patients (data not shown), suggesting that it 6 may not serve as an antigen for humoral immunity. We thus focused on NP and S-RBD. The 7 individual serum samples were then performed by serial dilutions to get optimal dilutions 8 ( Figure 1A ). Dilution of 1:50 was used for IgM and 1:450 for IgG. NP-and S-RBD-specific 9 IgM and IgG antibodies were both detected in the sera of newly discharged patients, 10 compared with healthy donor groups. Anti-SARS-CoV-2 IgG antibodies were also more 11 obviously observed than IgM in the follow-up patients (#9-14), when compared with healthy 12 donors ( Figure 1B ). In addition, values from the serum dilution curves were calculated to 13 determine the area under the curve (AUC) values. Compared to control sera, COVID-19 14 patient sera showed significantly higher AUC for NP-and S-RBD-specific IgG antibodies 15 ( Figure 1C ). Taken together, these findings indicate that COVID-19 patients mounted IgG 16 and IgM responses to SARS-CoV-2 proteins, especially NP and S-RBD, and also suggest 17 that infected patients could maintain their IgG amounts, at least for two weeks after 18 discharge. In addition, IgG isotypes was further tested in 14 patients and 6 controls. As shown in 20 Figure 1D , anti-NP and S-RBD IgG was mainly IgG1 isotype, and the newly discharged and 21 follow-up patients showed similarly amounts of anti-NP IgG1. Of interest, one patient (Pt#5) showed higher amounts of anti-NP IgG3, whereas anti-S-RBD IgG3 was detected in two 23 patients (Pt#4-5). However, we did not detect IgG2 to either NP or S-RBD proteins (data not 24 shown). Since the RBD of the S protein has been shown to bind to human angiotensin 1 converting enzyme 2 (ACE2) (Zhou et al., 2020) , the existence of antibodies against it may 2 suggest neutralization of SARS-CoV-2 infection. To assess this, we performed pseudovirus 3 particle-based neutralization assay, since there was a significantly positive correlation in the 4 neutralizing antibody titers between pseudovirus and SARS-CoV-2 ( Figure 2A ). As shown in 5 Figure 2B and 2C, patients #1, 2, 4, 5 and 8, all within the newly discharged group, had high 6 neutralizing antibody titers. These results demonstrate that most recently discharged 7 patients had strong humoral immunity to SARS-CoV-2. Among the follow-up patients, all had 8 neutralizing antibody titers with the exception of patient #9 being negative. As expected, 9 there was a significant correlation between neutralizing antibody titers and AUC of anti-S- To explore cellular immune responses to SARS-CoV-2, we isolated peripheral blood 16 monocytic cells (PBMCs) from the whole blood and phenotypically analyzed them by flow 17 cytometry ( Figure 3A ). We found that compared to newly discharged patients, there was a 18 trend towards an increased frequency of NK cells in the follow-up patients ( Figure 3B ). However, there was no significant difference in terms of the percentages of T cells among 20 those two groups and the healthy donors. To assess virus-specific cellular immunity, we then treated PBMCs with recombinant NP, 22 main protease and S-RBD, followed by IFN-γ ELISpot analysis. The results were considered 23 positive if there were at least 2-fold increase in the numbers of IFN-γ-secreting T cells in the 24 subject than in the healthy donors. As shown in Figure 3C , compared with healthy donors, 25 the numbers of IFN-γ-secreting NP-specific T cells in patients #1, 2, 4, 5 and 8 were much 26 higher than other patients, suggesting that they had developed SARS-CoV-2-specific T cell responses. Of note, patients #1, 2, 4, 5 and 8 developed both strong humoral and cellular 1 immune responses. Main protease-specific T cells were detected in patient #1, 2 and 5, 2 while patients # 1, 2, 4, 5, 6, 7 and 8 showed S-RBD-specific T cells. Although the numbers 3 of IFN-γ-secreting S-RBD specific T cells were much lower than those of NP-specific T cells, 4 they could be detected in more patients than those for other viral proteins. In the follow-up 5 patients, only patient #10 who showed lymphopenia before treatment still had a high number 6 of IFN-γ-secreting T cells in response to NP, main protease and S-RBD ( Figure 3C ), which 7 suggests that anti-viral T cells may not be maintained at high numbers in the PBMCs in the 8 recovered patients. More interestingly, when combining all 14 patients in our analysis, there 9 was a significant correlation between the neutralizing antibody titers and the numbers of NP- In this study, we characterized SARS-CoV-2-specific humoral and cellular immunity in 2 recovered patients. Both were detected in newly discharged patients. In addition, the 3 neutralizing antibody titers significantly correlated with the numbers of NP-specific T cells. These findings suggest both B and T cells participate in immune-mediated protection to viral 5 infection. Our work has thus provided a basis for further analysis of protective immunity to 6 SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe 7 cases. It has also implications in designing an effective vaccine to protect and treat SARS- In our study, production of S-RBD-specific antibodies were readily detected in recovered 10 patients. Moreover, we observed virus-neutralization activities in these recovered patients. Not surprisingly, a significant correlation between neutralizing antibody titers and AUC of 12 anti-S-RBD IgG, but not anti-NP IgG, was observed. Anti-S-RBD IgG might be useful in Nonetheless, in our study and the one mentioned above, most patients developed 27 measurable neutralization antibodies after infection, suggesting that the viral infection does 1 not curtail adaptive immunity. However, unlike the above-mentioned study, we did not find 2 any correlation between neutralizing antibody titers and patient's age, which could be due to 3 our small sample size. Our results thus need further confirmation in a large cohort of COVID-4 19 patients. In addition, our analysis could not differentiate CD4 + and CD8 + T cell responses, 5 due to the limitation in the amounts of PBMCs obtained and availability of instrumentation. The plasmid (pET28-N-6XHis) generated in this study will be made available on request 7 from the Lead Contact without restriction. The study did not generate any unique dataset or code. Committee at Tsinghua University. Informed consent was obtained from all subjects for 19 being included in the study. All patient data were anonymized before study inclusion. See 20 Table 1 for full patient information, including age, sex, and health status. Cell Lines 23 HuH-7 cells originally taken from a liver tumor in a Japanese male were cultured in DMEM 1 supplemented with 10% FBS. Cells were grown at 37 °C in a 5% CO2 setting. The OD value at 450 nm was calculated. Neutralizing antibody assay Pseudovirus expressing the SARS-CoV-2 S protein was produced as described previously 19 (Deng et al., 1997) . pNL43Luci and GP-pCAGGS were co-transfected into 293T cells. 48 Highlights: 1. SARS-CoV-2-specific antibodies are detected in COVID-19 convalescent subjects. 2. Most COVID-19 convalescent individuals have detectable neutralizing antibodies. 3. Cellular immune responses to SARS-CoV-2 are found in COVID-19 convalescent Human convalescence sera Notes: pt, patient; F, female; M, male; P, positive; N, negative; BT, before treatment; NA