key: cord-0748670-2rvfjhdw
authors: Hou, Hongyan; Zhang, Yandi; Tang, Guoxing; Luo, Ying; Liu, Wei; Cheng, Chang; Jiang, Yuhuan; Xiong, Zhigang; Wu, Shiji; Sun, Ziyong; Xu, Shabei; Fan, Xionglin; Wang, Feng
title: Immunological memory to SARS-CoV-2 in convalescent COVID-19 patients at one-year post-infection
date: 2021-09-15
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2021.09.008
sha: adddc6f7387961ef110533d6ac2b6f326788d796
doc_id: 748670
cord_uid: 2rvfjhdw

Background Understanding the complexities of immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to gain insights into the durability of protective immunity against reinfection. Objective To evaluate the immune memory to SARS-CoV-2 in convalescent patients with longer follow-up time. Methods SARS-CoV-2-specific humoral and cellular responses were assessed in convalescent coronavirus disease 2019 (COVID-19) patients at one-year post-infection. Results A total of 78 convalescent COVID-19 patients (26 moderate, 43 severe, and 9 critical) were recruited after one year of recovery. The positive rates of both anti-RBD and anti-N antibodies were 100%, whereas we did not observe a statistical difference in antibody levels among different severity groups. Accordingly, the prevalence of neutralizing antibodies (nAbs) reached 93.59% in convalescent patients. Although nAb titres displayed an increasing trend in convalescent patients with increased severity, the difference failed to achieve statistical significance. Notably, there was a significant correlation between nAb titres and anti-RBD levels. Interestingly, SARS-CoV-2-specific T cells could be robustly maintained in convalescent patients, and the number of them was positively correlated with both nAb titres and anti-RBD levels. Amplified SARS-CoV-2-specific CD4+ T cells mainly produced a single cytokine, accompanying with increased expression of exhaustion markers including PD-1, Tim-3, TIGIT, CTLA-4 and CD39, while the proportion of multifunctional cells was low. Conclusions Robust SARS-CoV-2-specific humoral and cellular responses are maintained in convalescent COVID-19 patients at one-year post-infection. However, the dysfunction of SARS-CoV-2-specific CD4+ T cells supports the notion that vaccination is needed in convalescent patients for preventing reinfection.

Coronavirus disease 2019 , the emerging infectious disease caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still the greatest threat to public health worldwide. 1 The production of SARS-CoV-2-specific antibodies, especially neutralizing antibodies (nAbs), is key for protecting against viral reinfection and provides insight into the design of vaccination strategies. 4, 5 Generally, SARS-CoV-2-specific IgM antibody level peaks at week 3 then declines, whereas IgG antibodies to spike protein can persist long-term, even beyond 6 months after infection. [6] [7] [8] [9] [10] However, nAb titres gradually decline after an initial peak in convalescent patients and most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. 11, 12 Notably, nAb titres are positively correlated with COVID-19 severity. [13] [14] [15] [16] It is noteworthy that, serum IgG antibodies to SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein correlate well with nAb titres, which suggests that commercially available anti-RBD antibodies can serve as useful surrogates for nAb testing. [16] [17] [18] Although previous studies have observed persistent humoral responses in convalescent patients, especially in those with severe disease for at least 6 months, how long nAbs will persist or whether they will provide protection from reinfection needs to be further studied.

and provide immunological memory that enables long-lasting protection, especially in individuals with negative or low titres of nAbs. [19] [20] [21] Emerging data indicate that SARS-CoV-2-specific CD8 + and CD4 + T cells targeting different viral proteins are detectable in up to 70% and 100% of convalescent individuals, respectively. [22] [23] [24] [25] More specifically, the membrane (M), S, and nucleocapsid (N) protein each account for 11%-27% of the total CD4 + T cell responses, with additional responses commonly targeting non-J o u r n a l P r e -p r o o f structural protein (nsp)-3, nsp-4, opening reading frame (ORF)-3a, and ORF-8, providing evidence that diversity of SARS-CoV-2 T cell responses is common in COVID-19 convalescent patients. 22 Notably, there is a strong correlation between the number of SARS-CoV-2-specific T cells and nAb titres. 5, 24 Although a recent study has found that SARS-CoV-2-specific T cell responses can be detected in convalescent patients at 6-7 months post-infection, 26 the duration of SARS-CoV-2-specific T cell memory, including the abundance, phenotype, and functional capacity, still needs to be further elucidated in patients with a longer recovery period.

A deep elucidation of immune memory to SARS-CoV-2 requires evaluation of its core elements, such as nAbs and CD4 + T cells. Understanding the complexities of immune memory to SARS-CoV-2 is key to gain insights into the likelihood of durability of protective immunity against reinfection. In this study, we assessed the SARS-CoV-2-specific anti-RBD and anti-N antibodies, nAbs, and CD4 + T cell responses simultaneously in convalescent COVID-19 cases, extending up to one year after infection. For the first time, our study provided evidence that although robust SARS-CoV-2-specific humoral and cellular responses were maintained in convalescent patients for as long as one year, the low titres of nAbs and exhausted function of SARS-CoV-2-specific CD4 + T cells indicated that vaccination was needed in convalescent patients for preventing reinfection. 

Blood samples were collected from study participants. SARS-CoV-2 antibodies

were measured using the quantitative Elecsys anti-RBD and semi-quantitative Elecsys anti-N (both measuring total immunoglobulin levels) on the Cobas e602 analyzer transformed before analysis.

Pseudovirus neutralization assay was performed as our previous study with minor modifications. 14 A full-length codon-optimized s gene of SARS-CoV-2 was first 

Heparinized blood samples were collected from study participants and peripheral 

The results are presented as mean ± standard deviation (SD), or as median with interquartile range (IQR) when appropriate. Continuous variables were compared with Mann-Whitney U-test or one-way ANOVA test. Fisher exact test was used for categorical data. Receiver operating characteristic curve analysis was performed to determine the best cutoff value of nAb titres for discriminating between convalescent COVID-19 patients and healthy controls. Spearman's rank correlation test for non-J o u r n a l P r e -p r o o f parametric data was employed to analyze the relationship between two factors.

Statistical significance was determined as p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

Statistical analyses were performed using SPSS version 19.0 (SPSS, Chicago, IL), GraphPad Prism 8.0 (San Diego, CA, USA). 

The current health status of all enrolled convalescent COVID-19 patients at one-

year post-infection was assessed by clinicians and the detailed information is shown in 

The reported in our previous study). 14 Although there were progressive increases in nAb titres in convalescent patients with increasing severity of COVID-19, we did not observe statistical difference among different groups (Fig 1, C) . Notably, there was a significant correlation between nAb titres and anti-RBD antibody levels in convalescent COVID-19 patients at one-year post-infection, but anti-N antibody levels did not correlate with nAb titres, suggesting that anti-RBD antibody levels might be predictive of serum neutralization capabilities in COVID-19 patients (Fig 1, D and E) .

To gain a comprehensive insight into the immune memory to SARS-CoV-2, the frequency of viral-specific T cells was detected by using SARS-CoV-2-specific ELISPOT assay (Fig 2, A) . Surprisingly, after SARS-CoV-2 peptide pool stimulation, SARS-CoV-2-specific T cells were noted in 100% (10/10) of convalescent COVID-19

patients after one year of recovery. Unlike nAbs, the number of SARS-CoV-2-specific T cells in severe convalescent patients was higher than that in moderate convalescent patients (Fig 2, B) . Notably, the frequency of SARS-CoV-2-specific T cells was significantly positively correlated with nAb titres and anti-RBD antibody levels (Fig 2, C and D). These data confirmed the robust persistence of SARS-CoV-2-specific T cell responses in convalescent COVID-19 patients at one-year post-infection.

Upon peptide pool stimulation, IFN- + SARS-CoV-2-specific CD4 + T cells were J o u r n a l P r e -p r o o f predominant effector memory cells (non-CD45RA + CCR7 + cells) (Fig 3, A) . After stimulation, amplified CD4 + T cells mainly produced a single cytokine (IFN-, TNF-, or IL-2), whereas the percentage of multifunctional CD4 + T cells (IFN- + TNF- + or IFN- + TNF- + IL-2 + ) was obviously lower than that of single cytokine-producing CD4 + T cells (Fig 3, B and C) . Considering that SARS-CoV-2-specific CD4 + T cells mainly The subsets of SARS-CoV-2-specific CD4 + T cells were also determined according to the production of intracellular cytokines including IFN- (Th1), IL-4 (Th2), and IL-17 (Th17) (Fig 5, A) . 29 After peptide pool stimulation, the percentages of IFN- + CD4 + Th1 cells were significantly higher than those of IL-17 + CD4 + Th17 cells. However, we almost did not observe the differentiation of IL-4 + CD4 + Th2 cells under peptide pool stimulation (Fig 5, B) . Thus, SARS-CoV-2-specific CD4 + T cells were mainly differentiated into Th1 and Th17 cells, but not Th2 cells.

Moreover, after peptide pool stimulation, the percentage of IFN--and TNF-producing CD4 + T cells were positively and negatively correlated with anti-RBD antibody levels, respectively. However, we did not observe a statistical correlation between the percentages of other cytokine-producing CD4 + T cells and antibody levels ( Fig 6) . 

Furthermore, given that type I IFN immunity is essential for protective immunity to respiratory infection with SARS-CoV-2, a very recent study has indicated that autoantibodies neutralizing type I IFNs predate SARS-CoV-2 infection and cause critical COVID-19, especially in the elderly. 34 Thus, it is noteworthy that unlike nAbs, some types of antibodies in peripheral blood may not provide protection from infection, but conversely exacerbate illness.

In addition to nAbs, viral-specific T cells also contribute to clearance of the acute infection. 35 The antigen-specific immunity depends on both the frequency and function of memory cells. We therefore further assessed the cytokine secretion ability and phenotypes of SARS-CoV-2-specific CD4 + T cells. To our surprise, upon peptide pool stimulation, SARS-CoV-2-specific CD4 + T cells mainly produced a single cytokine, such as IFN-, TNF- and IL-2, which is inconsistent with previous study that amplified CD4 + T cells often showed multi-functionality (expressing both IFN- and TNF-) in patients with short-term recovery. 28 The different sampling intervals might cause such disparity. These data suggested that SARS-CoV-2-specific CD4 + T cells Regarding PD-1 expression, a study found that PD-1-expressing SARS-CoV-2specific CD8 + T cells were not exhausted, but functional in patients with This is in accordance with our previous study showing that increased expression of PD-1 on CD4 + T cells is correlated with higher cytokine secretion capability after PMA/ionomycin stimulation. 39 However, this study demonstrated that PD-1-expressing SARS-CoV-2-specific CD4 + T cells were exhausted but not multifunctional, and this discrepancy may be due to different detection periods. Previous studies assessed the 

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J o u r n a l P r e -p r o o f In-house diluted leftover serum sample with high antibody levels, allowing for inter-and intra lot comparison (determination of the coefficient of variation). # Manufacturer's positive and negative quality controls (Roche SARS-Cov2 S PreciControl) that need to be respectively between 7.21-13.4 U/ml and below 0.4 U/ml, in order for the run to be carried out.