key: cord-0911267-1npph2gw
authors: Yao, Lin; Wang, Guo-Lin; Shen, Yuan; Wang, Zhuang-Ye; Zhan, Bing-Dong; Duan, Li-Jun; Lu, Bing; Shi, Chao; Gao, Yu-Meng; Peng, Hong-Hong; Wang, Guo-Qiang; Wang, Dong-Mei; Jiang, Ming-Dong; Cao, Guo-Ping; Ma, Mai-Juan
title: Persistence of Antibody and Cellular Immune Responses in COVID-19 patients over Nine Months after Infection
date: 2021-05-12
journal: J Infect Dis
DOI: 10.1093/infdis/jiab255
sha: 68a63e29d89314041abc746f96d1619a6b56b2cb
doc_id: 911267
cord_uid: 1npph2gw

BACKGROUND: The duration of humoral and T and cell response after the infection of SARS-CoV-2 remains unclear. METHODS: We performed a cross-sectional study to assess the virus-specific antibody and memory T and B cell responses in COVID-19 patients up to 343 days after infection. Neutralizing antibodies and antibodies against the receptor-binding domain, spike, and nucleoprotein of SARS-CoV-2 were measured. Virus-specific memory T and B cell responses were analyzed. RESULTS: We enrolled 59 COVID-19 patients, including 38 moderate, 16 mild, and five asymptomatic patients; 31 (52.5%) were men, and 28 (47.5%) were women. The median age was 41 (interquartile range [IQR]: 30–55). The median day from symptom onset to enrollment was 317 days (range 257 to 343 days). We found that approximately 90% of patients still have detectable IgG antibodies against spike and nucleocapsid proteins and neutralizing antibodies against pseudovirus, whereas ~60% of patients had detectable IgG antibodies against receptor binding domain and surrogate virus-neutralizing antibodies. SARS-CoV-2-specific IgG (+) memory B cell and IFN-γ secreting T cell responses were detectable in over 70% of patients. CONCLUSIONS: SARS-CoV-2-specific immune memory response persists in most patients nearly one year after infection, which provides a promising sign for prevention from reinfection and vaccination strategy.

Lin Yao 1, a , Guo-Lin Wang 1 

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus disease (COVID- 19 ) agent, has caused the pandemic worldwide [1] [2] [3] . As of February 2, 2021, more than 100 million confirmed cases of SARS-CoV-2 had been reported, with more than 2 million deaths [4] . Effective vaccines are vital for controlling the pandemic. Therefore, understanding the long-term immunological memory response to SARS-CoV-2 after nature infection is critical for developing and implementing a SARS-CoV-2 vaccine. Recent studies showed most patients persist virus-specific antibody response 6-8 months after infection but displayed a waning trend of humoral immunity in patients over time [5] [6] [7] [8] [9] [10] [11] [12] . SARS-CoV-2-specific memory CD8 + and CD4 + T cells were detected in most patients but declined with a half-life of 3-5 months [13], whereas virus-specific memory B cells (MBCs) continued to rise or keep unchanged 5-6 months after the infection [7, 8, 13, 14] . However, the antibody and memory T and B cell response beyond eight months or nearly one year after the infection is unclear.

Therefore, in this study, we conducted a cross-sectional study of 59 patients up to 343 days after infection and assessed the virus-specific antibody and memory T cells and B cells.

Between December 7 and 30, 2020, patients who recovered from the COVID-19 with SARS-CoV-2 infection in Jiangsu, Shandong, and Zhejiang Provinces, China, were invited to participate in this study. All of the patients were laboratory-confirmed positive for SARS-CoV-2 by real-time reverse transcription-polymerase chain reaction (rRT-PCR) results. Each enrolled patient provided a 3 ml blood sample for serum isolation and an additional 5 ml blood sample for peripheral blood mononuclear cells (PBMCs) isolation. Demographics and clinical characteristics of patients were collected upon enrollment. Thirty age-and sex-matched healthy controls (HCs) enrolled before the pandemic were used as control. Each patient signed informed consent. The study protocol was A c c e p t e d M a n u s c r i p t 5 at 450nm and 630nm. Endpoint antibody titers were calculated by a fitted curve (4 parameter log regression), and the three times the average value of HCs was used as the detection threshold.

Serum IgG to nucleocapsid (N) protein of SARS-CoV-2 was semiquantitatively measured by ELISA A c c e p t e d M a n u s c r i p t

The SARS-CoV-2 pVNT was performed as described previously [4] . 

To assess B cells secreting IgG antibodies specific for SARS-CoV-2 RBD and cells secreting IgG 

One-way ANOVA with LSD post-hoc testing (normal distribution) or Kruskal-Wallis test with false discovery rate method (non-normal distribution) was used for multiple group comparisons. Mann-Whitney U test was used to compare the difference between the two groups. Spearman correlations analyses were used to determine associations between analyzed parameters. All statistical analyses were performed using GraphPad Prism (version 8.4.2, La Jolla, California USA), and all statistical tests were 2-sided with a significance level of 0.05.

A c c e p t e d M a n u s c r i p t 8

We enrolled 59 recovered COVID-19 patients with a time interval of 317 days (range 257 to 343 days, defined 9-11 months after infection) from symptom onset to sampling. Of the 59 patients, 38 were moderate, 16 were mild, and five were asymptomatic ( 

We first assessed the SARS-CoV-2 RBD binding IgG antibody using ELISA and found that 55.9% IgG titer (>500). Although our focus was on the S protein, we also semiquantitatively measured the antibody response to the N protein of SARS-CoV-2 since this is the antigen targeted by multiple commercial assays. We observed anti-N IgG antibodies are persistently high in recovered patients, and 98.3% (58/59) of patients (100% for asymptomatic/mild and 97.4% for moderate patients were still positive ( Figure 1C ). As expected, in the serum of HCs, we observed a minimal reactivity of anti-S or anti-RBD IgG antibodies ( Figure 1A and 1B), whereas one serum was detected positive for anti-IgG antibodies ( Figure 1C ).

A c c e p t e d M a n u s c r i p t 9 To determine whether patients maintain neutralizing antibodies, we detected SARS-CoV-2 neutralization indirectly using a cell-free assay of RBD-ACE2 binding inhibition by sVNT and directly pVNT. sVNT assays showed that 66.1% (39/59) of patients (71.4% for asymptomatic/mild and 63.2% for moderate patients) had antibody to inhibit RBD binding to ACE2 ( Figure 1D ), and 44% (26/59) patients displayed > 50% inhibit rate to ACE2 ( Figure   1D ). Further pVNT assay revealed that 89.8% (53/59) patients (21/21 asymptomatic/mild and 32/38 moderate patients) had detectable NAb ( Figure 1E ). The geometric mean titer Figure 1E ). We found that IgG antibodies were strongly correlated with each other and neutralizing inhibition rate, whereas they were moderately correlated with pesudovirus-based NAb titer (Supplementary Figure   1 ).

Although no significant differences were observed for antibody response between asymptomatic/mild and moderate patients, moderate patients tended to a slightly low positive antibody response rate. The fact that most moderate patients (median day of 320, IQR 313-331) had a longer time interval from symptom onset to sampling than those asymptomatic/mild patients (median day of 312, IQR 306-318, p = 0.0025). Further analysis showed that the age, sex, and underlying medical conditions were not associated with antibody responses except for a higher anti-S IgG titer (Supplementary Figure 2) .

We further assessed virus-specific memory B and T cell responses among these patients using ELISpot assay (Figure 2A and 2D A c c e p t e d M a n u s c r i p t 10 significantly higher than in HCs ( Figure 2D and 2E ). In addition, 2 of 20 (6.7%) (2/30) HCs had detectable IFN-γ-secreting T cells ( Figure 2E ). There was correlation between SARS-CoV-2 specific

MBCs response and anti-RBD IgG (r = 0.350, p = 0.007), anti-S IgG (r = 0.330, p = 0.010), and NAbinhibition rate (r=0.456, p < 0.001) but not anti-N IgG ( Figure 2F ). However, no significant correlations were observed between the SFC of SARS-CoV-2 specific T cells secreting IFN-γ and antibody response (Supplementary Figure 3) . Further analysis showed that patients older than 60 years old had fewer memory B cells secreting total IgG and RBD-specific IgG than patients under 60 years old (Supplementary Figure 4) .

Recent studies have shown that most patients had detectable SARS-CoV-2 antibody responses 6-8 months after infection [6, 12, [16] [17] [18] [19] . This study evaluated SARS-CoV-2 specific antibody and cellular immune responses in patients up to 11 months after infection. Our data reveal that approximately 70% of COVID-19 patients maintain anti-RBD and -S IgG, ACE2-RBD inhibition rate, and NAb at least 9-11 months after infection. Moreover, we observed that pseudovirus neutralizing antibodies and antibodies against the N and S proteins are longer-lived than those against RBD. Although antibodies to RBD of SARS-CoV-2 S protein accounted for the majority of IgG responses, antibodies to other epitopes such as the N-terminal domain and S2 subunit of S protein also contribute to the IgG response [20] . A previous study also showed that the combination of antibodies to other epitopes of S protein and antibodies against RBD could more effectively block the virus from invading host cells [21] . Therefore, the antibody titer and positivity rates of anti-RBD IgG and ACE2-RBD inhibition might be lower than the NAb and anti-S IgG response. We also observed relatively high (~98%) positive detection of anti-N IgG among patients compared to other antibodies. In human SARS-CoV-1 infection, antibodies against the SARS-CoV-1 N protein are abundant and longer-lived than other viral components such as the S, M, and envelope proteins [22] . In another study, antibodies against S1 or RBD persisted longer than antibodies against N protein in the sera of SARS survivors 17 years after infection [23] . Although this phenomenon's biological significance and mechanistic characterization are beyond this study's scope, further investigation is warranted.

A c c e p t e d M a n u s c r i p t 11 Whether primary infection with SARS-CoV-2 protects individuals from reinfection and how long has yet to be established. Patients with SARS-CoV-2 reinfection are still rare but on the rise [24] .

Reinfections may imply that immunity against SARS-CoV-2 may be weak and decay relatively quickly, with implications not just for the risks facing recovered patients but also for how long future vaccines might protect people [25] . Notably, several reported cases with SARS-CoV-2 reinfection displayed a low level or without producing the antibody response after prior infection [26] [27] [28] [29] [30] , which may hamper a more effective response to the second time around. In contrast, recent studies have shown that the presence of antibodies to SARS-CoV-2 was associated with a significantly reduced risk of SARS-CoV-2 reinfection for six to seven months after prior infection [31, 32] . Moreover, prior infection also reduces the risk of asymptomatic infection and likely to be protective against severe disease or symptomatic infection [31, 32] . These data provide clues on the protection of humoral immune response against reinfection. Collectively, sustained humoral immunity after infection might help apply vaccines for reliable prevention of SARS-CoV-2 transmission, patients who became seronegative for antibody response may be at higher risk of reinfection when they meet the next exposure compared to seropositive individuals.

While most patients had detectable virus-specific antibodies 9-11 months after infection, the longevity of virus-specific memory T cells and MBCs is still unresolved. Previous studies have shown that memory CD4 + and CD8 + T cells can be detected in 70%-100% early convalescent COVID-19 patients [33] [34] [35] . In addition, broad and strong memory CD4 + and CD8 + T cells can be found in >90% convalescent COVID-19 patients [36, 37] . Recent [8, 14] . Moreover, more abundant S-specific MBCs were detected in approximately 80% of patients at six months compared to one month after infection [13] , and Sspecific and RBD-specific MBCs can even be respectively detected in 78% and 100% patients for up to eight months post-symptom onset [40, 41] . Consistent with the above evidence, we found that over 70% of patients had detectable both virus-specific T cells producing IFN-γ and RBD-specific IgG There are several limitations of this study. One of the major limitations of this study is a crosssectional study that limited the observation of the dynamic changes of antibody or immune cells over time that limits generalization. Another limitation is the relatively small number of patients and the paucity of severe patients in this study. Because the most severe patients in the field of the study conducted were elderly, and they and their family members had a very low willingness to participate in the study. However, previous studies have shown that severe patients had a higher antibody titer than other disease conditions [42, 43] .

In summary, detectable humoral and cellular immunity can be found in most of the patients 9-11 months after infection with SARS-CoV-2 offers insights into the long-term immune response to SARS- 6  16  19  25  57  46  29  15  43  11  18  35  32  26  24  40  56  4  14  54  42  5  20  12  1  13  9  50  21  48  53  36  10  7  17  45  47  55  27  41  58  3  8  22  23  28  30  31  33  34  37  38  39  44  49  51  52  59   0   1000   500   6  2  19  20  57  40  50  46  38  25  43  32  9  37  56  26  15  11  24  21  42  54  49  7  35  45  29  12  18  39  14  36  16  41  1  44  47  4  13  23  10  33  17  22  48  31  5  28  30  52  8  59  34  3  27  55  53  58  51   0   6000   500  66.7   43  35  46  19  40  57  6  32  15  2  16  25  56  26  54  18  42  29  11  50  24  38  36  41  9  48  12  7  14  44  39  47  52  13  21  20  10  4  49  31  28  45  53  1  34  55  37  22  58  33  23  8  30  7  2  5  17  59 6  33  20  26  40  15  35  38  54  9  23  24  11  7  14  43  51  18  4  13  57  21  28  27  10  34  19  29  2  48  37  36  12  52  41  49  47  39  50  8  25  56  5  1  16  3  22  45  55  17  30  44  53  58 46  43  32  29  2  56  19  57  18  11  40  26  24  7  15  10  54  14  50  39  42  35  41  16  48  20  12  45  4  47  52  21  49  38  13  44  5  36  59  1  22  23  53  30  33  28  37  17  55  34  8  31  58  27  3 

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We thank all patients for their participation in this study. 

We declare no competing interests.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t