key: cord-329129-t84pu00z
authors: Zuo, J; Dowell, A; Pearce, H; Verma, K; Long, HM; Begum, J; Aiano, F; Amin-Chowdhury, Z; Hallis, B; Stapley, L; Borrow, R; Linley, E; Ahmad, S; Parker, B; Horsley, A; Amirthalingam, G; Brown, K; Ramsay, ME; Ladhani, S; Moss, P
title: Robust SARS-CoV-2-specific T-cell immunity is maintained at 6 months following primary infection
date: 2020-11-02
journal: bioRxiv
DOI: 10.1101/2020.11.01.362319
sha: 
doc_id: 329129
cord_uid: t84pu00z

The immune response to SARS-CoV-2 is critical in both controlling primary infection and preventing re-infection. However, there is concern that immune responses following natural infection may not be sustained and that this may predispose to recurrent infection. We analysed the magnitude and phenotype of the SARS-CoV-2 cellular immune response in 100 donors at six months following primary infection and related this to the profile of antibody level against spike, nucleoprotein and RBD over the previous six months. T-cell immune responses to SARS-CoV-2 were present by ELISPOT and/or ICS analysis in all donors and are characterised by predominant CD4+ T cell responses with strong IL-2 cytokine expression. Median T-cell responses were 50% higher in donors who had experienced an initial symptomatic infection indicating that the severity of primary infection establishes a ‘setpoint’ for cellular immunity that lasts for at least 6 months. The T-cell responses to both spike and nucleoprotein/membrane proteins were strongly correlated with the peak antibody level against each protein. The rate of decline in antibody level varied between individuals and higher levels of nucleoprotein-specific T cells were associated with preservation of NP-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T-cell responses are retained at six months following infection although the magnitude of this response is related to the clinical features of primary infection.

The SARS-CoV-2 pandemic has led to over 1 million deaths to date and there is an urgent need for an effective vaccine (1) . There is considerable interest in understanding how adaptive immune responses act to control acute infection and provide protection from reinfection.

Antibody responses against SARS-CoV-2 are characterised by responses against a range of viral proteins, including the spike, nucleocapsid and membrane proteins. A number of studies, however, have shown that the level of this antibody response declines over time and may even lead to loss of detectable virus-specific antibodies in a substantial proportion of individuals (2, 3) . Information derived from study of immunity to related viruses such as SARS-CoV-1 and MERS (4) has shown that cellular immune responses against these viruses are maintained for much longer periods of time compared to antibody responses (5, 6) . This has led to the hope that cellular responses to SARS-CoV-2 will similarly be of more prolonged duration (7, 8) .

Studies to date have shown that virus-specific cellular responses develop in virtually all patients with confirmed SARS-CoV-2 infection (9) . These responses remain detectable for several weeks following infection but it is currently unknown how they are maintained thereafter (10) . In this study we characterised SARS-CoV-2-specific T cell immune responses in a cohort of 100 donors at 6-months post-infection.

Blood samples were obtained from 100 convalescent donors at 6 months following initial SARS-CoV-2 infection in March-April 2020. Among the 100 donors, 77 (77%) were female and 23 (23%) were male with a median age of 41.5 years (22-65 years) . None of the donors required hospitalisation at any time during the course of the study. 56 (45 female and 11 male) of the 100 donors who experienced clinical symptoms of respiratory illness were grouped as "symptomatic" and 44 (32 female and 12 male) who did not experience any respiratory illness were grouped as "asymptomatic". There was no significant difference between the median age of the symptomatic (42.5 (23-61) years) and asymptomatic donors (40 (23-65) years).

Interferon gamma (IFN-g) ELISPOT analysis was used to determine the magnitude of the global SARS-CoV-2-specific T cell response. Peptide pools from a range of viral proteins, including spike, nucleoprotein and membrane protein, were used to stimulate fresh PBMC and the magnitude of the global SARS-CoV-2-specific T-cell response was determined. Median ELISPOT responses against the Spike glycoprotein (Spike); Nucleoprotein and Membrane (N/M); and ORF3a, ORF10, NSP8, NSP7A/b (Accessory) peptide pools were measured at 1 in 10,000 (0.010%), 12,500 (0.008%) and 66,666 (0.0015%) PBMC respectively ( Figure 1A ). Using the pre-2020 healthy donor PBMCs to set the cut-off point, 90 of 95 donors (95%) demonstrated a SARS-CoV-2-specific T-cell response to at least one protein with a median T cell immunity to SARS-Cov-2 at 6 months post infection 5 total value of 200 cells per million PBMC (1 in 5000) ( Figure 1A ). Eighteen donors did not have a demonstrable cellular response to Spike and no response to the N/M pool was seen in 9

individuals. No detectable response to any protein tested was seen in 5 donors by ELISPOT assay although all these donors responded by parallel intracellular cytokine analysis ( Figure   1B ).

Considerable heterogeneity was observed in relation to the magnitude of this response. The global and peptide-specific responses were then assessed in relation to the clinical features at the time of primary infection. Importantly, median aggregate ELISPOT responses were 50% higher in donors who had initially demonstrated symptomatic disease compared to those with asymptomatic infection (Figure 2A ). This profile was consistent against both spike and aggregate N/M proteins where values were 42% and 55% higher respectively in donors with initial symptomatic infection ( Figure 2B ). No association was seen between ELISPOT response and donor age.

Intracellular cytokine analysis was then utilised to assess the specificity and pattern of cytokine production from SARS-CoV-2-specific CD4+ and CD8+ T-cells in 100 donors. Virusspecific cytokine responses were seen in 96 people, including the 5 individuals that had been negative by ELISPOT analysis ( Figure 3A ). Interestingly, CD4+ virus-specific T cell responses were twice as frequent as CD8+ responses at this six-month time point (0.025% of CD4+ pool vs 0.012% of CD8+ pool respectively) ( Figure 3B ). In particular, mean CD4+ responses against spike and non-spike (N/M/Accessory) proteins were measured at 0.009% and 0.016% of the CD4+ repertoire respectively whilst corresponding values for CD8+ cells were 0.0045% and T cell immunity to SARS-Cov-2 at 6 months post infection 6 0.0078% ( Figure 3B ). No differences were observed in the virus-specific CD4:CD8 ratio in relation to demographic factors such as age, symptomatic disease or gender.

As expected, the pattern of cytokine production differed between the CD4+ and CD8+ subsets ( Figure 4A, B) . It was noteworthy that IL-2 responses were dominant within CD4+ subset ( Figure 4B ). Of note, the pattern of cytokine production by virus-specific CD4+ T cells was dependent on antigenic specificity. Single IFN-g, single IL-2 and dual positive IL-2+IFN-g+ T cells comprised 0.0016%, 0.0051% and 0.0027% of the spike-specific CD4+ T cell response respectively, compared to 0.0017%, 0.0105% and 0.0031% of the non-spike-specific repertoire ( Figure 4C ). The magnitude of CD4+ T cell responses against spike and non-spike proteins within each individual was strongly correlated ( Figure 4D ). However, this association was less marked for the CD8+ subset where responses were dominant against non-spike proteins ( Figure 4D ).

We next assessed how the magnitude, phenotype and cytokine profile of the virus-specific cellular immune response at six months correlated with the prospective profile of antibody production in the six months since infection. Antibody levels against both the Spike glycoprotein and nucleoprotein were available at serial time points from all donors ( Figure   5A ). These were used to define both the peak value of antibody level against each protein and the rate of decline in antibody level over the subsequent two months. Peak antibody levels against both spike and nucleoprotein were observed typically at the second month of sampling ( Figure 5A ). Antibody levels fell by approximately 50% during the two months after peak level but stabilised somewhat thereafter although spike-specific responses continued to decline ( Figure 5A ).

Interestingly, the magnitude of the T cell ELISPOT response at 6 months against the spike protein was strongly correlated with magnitude of the peak antibody level against both spike protein and the RBD domain ( Figure 5B ). A similar correlation was observed between the cellular response to the N/M pool and the peak level of N-specific antibody ( Figure 5B ).

The rate of antibody decline was then assessed in relation to the profile of the cellular immune response at 6 months. Relative preservation of the N-specific antibody response was seen in donors with stronger N and M-specific T cell responses at six months suggesting the cellular responses may act to support antibody production ( Figure 5C ). However, no such association was observed in relation to spike-specific responses.

Finally, we also assessed the profile of CXCR5 expression on virus-specific T cells and related this to the pattern of stability of the virus-specific antibody response as positive correlations have been observed previously in HIV infection (11) . High numbers of circulating Tfh CD4+ T cells have been seen in severe acute infection (12) but at 6 months CXCR5 was expressed on only 7% of virus-specific CD4+ T cells and no correlation was observed with the profile of Ab level following infection.

The magnitude and quality of the immune memory response to SARS-CoV-2 will be critical in preventing reinfection. Here we undertook, to our knowledge, the first assessment of the SARS-CoV-2-specific T cell immune response at six months following primary infection in a unique cohort of healthy adults with asymptomatic or mild-to-moderate COVID-19.

The major finding was that virus-specific T cells were detectable in all donors at this extended follow-up period. A high prevalence of detectable T-cell immunity has been described in studies performed at earlier time points after infection and our findings indicate that robust memory is maintained for at least 6 months. Approximately 1 in 4000 PBMC were SARS-CoV-2-specific which is broadly comparable to findings within the first three months after infection. These values are lower than typical responses against persistent herpesviruses (13) but comparable to those against acute respiratory viruses, including SARS-CoV (14, 15) .

The magnitude of the T cell response was heterogeneous and may reflect the reports of remarkable diversity in the profile of the T cell immune response during acute infection (16) .

A striking feature was that the magnitude of cellular immunity was considerably higher in donors who had experienced symptomatic disease. Indeed, median ELISPOT responses were 50% higher within this group and demonstrate that the initial relative 'setpoint' of cellular immunity established following acute infection is maintained for at least 6 months. A similar pattern has been observed within the first few weeks following acute SARS-CoV-2 infection in patients recovering from severe versus mild disease (17) and also in patients after SARS infection (30) . This is likely to reflect a response to higher viral loads and inflammatory mediators during acute infection (18, 19) although it is also possible that an elevated adaptive immune response during primary infection can itself act as a determinant of the clinical phenotype (20) . Cellular responses have a direct protective effect against severe coronavirus infection (21) and also support antibody production. Indeed, cytokine analysis showed that the CD4+IL-2+ subset was most significantly elevated in the symptomatic group. The finding of lower levels of T cell immunity in asymptomatic donors at 6 months after infection might potentially add to concerns that this group may be more susceptible to later re-infection. However, it is also possible that the quality of the T cell response at the time of initial infection was sufficient to provide clinical protection. The relative susceptibility of patients with initial asymptomatic disease to episodes of re-infection, either clinically silent or symptomatic, will therefore need to be assessed over time.

It was noteworthy that CD4 T cells responses against SARS-CoV-2 outnumbered CD8 effector cells by ratio of 2 to 1. Again, a similar pattern has been demonstrated at earlier time points after SARS-CoV-2 infection and may reflect high levels of viral protein uptake by antigenpresenting cells and cross presentation to the CD4+ positive T-cell pool or preferential expansion of CD4+ T cells (22) . Furthermore, cytokine analysis showed that IL-2 was the major cytokine produced by virus-specific CD4+ cells, indicating a proliferative potential which may auger well for long-term immune memory (23) . IFN-g responses are broadly equivalent to IL-2 at early time points after infection (24) but the profile at 6 months suggests that the relative proportion of Th1 effector cells decreases over time or they revert to central memory state (25) . Polyfunctional T cells are typically associated with superior pathogen control (26) and studies on SARS-CoV-2 infections have revealed decreased cytokine functionality in patients with severe disease (17) . The majority of CD4+ T cells at six months expressed only a single cytokine and production of three or four cytokines was observed in <15% of cells. These results were consistent with comprehensive analysis of the cytokine profile released by SARS-CoV-2 specific T cells in supernatants from the ELISPOT assay which showed that IL-2 was consistently the dominant cytokine. Interestingly, low levels of IL-10 were released in response to all the peptide pools and as IL-10 production has been reported by subsets of murine Influenza and Coronavirus specific T cells (27, 28) these represent an interesting population of cells for future investigation. Low and variable concentrations of IL-4 and TNFα were also detected.

Of note, the pattern of cytokine production by CD4+ T cells varies with protein specificity, as seen in earlier reports (17) . Single IL-2 or IFN-g producing cells were predominant against both spike and structural proteins but the former population was significantly greater in the CD4+ response against non-spike proteins, indicating that a retained Th1 effector profile is more common within spike-specific T cell pool. These dual-positive populations are associated with elite immunological control of HIV infection and indicate that spike-specific T cells preferentially retain characteristics of both effector function and proliferative potential in vivo.

The expression of CXCR5 on CD4+ T cells has been correlated with the magnitude and persistence of humoral immunity in the setting of HIV infection (29) . We, however, observed that CXCR5 was expressed on only 7% of virus-specific CD4+ T cells suggesting that circulating virus-specific follicular helper cells are not sustained after infection and no clear relationship was noted between CXCR5 expression and either the magnitude or the rate of decline of SARS-CoV-2-specific antibody level. Findings in acute infection have also failed to correlate cTFh frequencies with the plasmablast response and suggest that non-CXCR5+ CD4+ T cell help may also operate (16).

One of the valuable features of our cohort was the availability of monthly antibody levels against the spike and nucleoproteins in the first six months after infection. The finding that that higher T cell responses at 6 months against N/M proteins correlated with slower decline in N-specific antibody levels indicates that vaccine approaches that elicit strong cellular immune responses against this protein are likely to be valuable for sustaining stable antibody responses. In contrast, T-cell responses against Spike were not related to the rate of decline of antibodies against that protein. Nevertheless, spike protein-specific cellular responses were present in >80% of individuals at 6 months after mild to moderate infection and are also recognised as an immunodominant protein following SAR-CoV-1 infection (30) . Spike glycoprotein is the major immunogen used in current vaccine trials and these findings indicate that strong and sustained spike-specific T-cell immunity is likely to be required to sustain immune protection and should be assessed in analysis of optimal vaccine strategies. Our finding that T cell responses against M/N proteins are equally as high as Spike responses at 6months after natural infection suggest that these proteins could also represent valuable components of future vaccine strategies.

Our findings demonstrate that robust cellular immunity against SARS-CoV-2 is likely to be present within the great majority of adults at six months following asymptomatic and mildto-moderate infection. These features are encouraging in relation to the longevity of cellular immunity against this novel virus and are likely to contribute to the relatively low rates of reinfection that have been observed to date (31) . Further studies will be required to assess how these immune responses are maintained over the longer term. Mean spot counts for negative control wells were subtracted from the mean of test wells to generate normalised readings, these are presented as Spot Forming Cells per million input PBMC (SFC/10 6 PBMC).

Freshly isolated PBMC were rested overnight prior to the assay. 1.5x10 6 

Following overnight peptide stimulation in ELISPOT assays 50ul of supernatant was removed and combined from two duplicate wells and cryopreserved at -80°C. Supernatant from eleven donors responding in the ELISPOT assay were profiled using a 12-plex Legendplex T Helper Cytokine Panel Version 2 (Biolegend) following the manufactures instructions. Cytokine beads were analysed on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, US). Data was analysed with Legendplex Software (Biolegend) and the average cytokine level determined from two duplicate samples.

Statistical analysis was performed with GraphPad Prism 8. A Mann-Whitney 2-tailed U test was used to compare variables between two groups, a Wilcoxon matched-pairs signed rank test was used to compare paired non-parametric data, and a Friedman test with Dunn's multiple comparisons test was used to compare non-parametric data between more than 2 groups. Correlations were performed via Spearman's rank correlation coefficient. Two-way ANOVA with Dunnett multiple comparisons test was used to determine significance of cytokine profile data. Statistical significance was determined as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. 

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This work was partly funded by UKRI/NIHR through the UK Coronavirus Immunology