key: cord-0751501-wpyalhl2
authors: Clarke, C. L.; Prendecki, M.; Dhutia, A.; Edwards, C.; Prout, V.; Lightstone, L.; Parker, E.; Marchesin, F.; Griffith, M.; Charif, R.; Pickard, G.; Cox, A.; McClure, M.; Tedder, R.; Randell, P.; Greathead, L.; Guckian, M.; McAdoo, S. P.; Kelleher, P.; Willicombe, M.
title: Longevity of SARS-CoV-2 immune responses in haemodialysis patients and protection against reinfection
date: 2021-01-26
journal: nan
DOI: 10.1101/2021.01.22.21249865
sha: 3d75906495c7a6637944c7e90a667ba8a8f76237
doc_id: 751501
cord_uid: wpyalhl2

Background Patients with end stage kidney disease (ESKD) receiving in-centre haemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, ICHD patients frequently develop serological evidence of infection, even with asymptomatic disease. The aim of this study is to investigate the durability and functionality of immune responses to SARS-CoV-2 infection in ICHD patients. Methods Three hundred and fifty-six ICHD patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies. Patients who became seronegative at 6 months were investigated for SARS-CoV-2 specific T-cell responses. Results One hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at Time 0, of which 127(98.4%) also had detectable anti-RBD. At 6 months, of 111 patients tested, 71(64.0%) and 97 (87.4%) remained anti-NP and anti-RBD seropositive respectively, p<0.001. For patients who retained antibody, both anti-NP and anti-RBD levels reduced significantly after 6 months. Ten patients who were anti-NP and anti-RBD seropositive at Time 0, had no detectable antibody at 6 months; of which 8 were found to have SARS-CoV-2 antigen specific T cell responses. Independent of antibody status at 6 months, patients with serological evidence of SARS-CoV-2 antibodies at Time 0, were at significantly lower risk of being diagnosed with infection Conclusions ICHD patients mount durable immune responses 6 months post SARS-CoV-2 infection, with <3% of patients showing no evidence of humoral or cellular immunity. These immune responses are associated with a reduced risk of subsequent reinfection.

Efficacy results from several SARS-CoV-2 vaccine trials was welcome news at the end of 2020, as the roll out of effective vaccination programmes set to mark the beginning of the end of the pandemic(1-3). The Moderna (mRNA-1273), Pfizer/BioNTech (BNT162b2 mRNA) and Oxford/AstraZeneca (ChAdOx1 nCoV- 19) vaccines have all been shown to induce robust humoral and cellular immune responses against the spike protein of the SARS-CoV-2 virus, which importantly protect individuals from risk of subsequent infection (4, 5) . However, given the logistical issues associated with supply, distribution and administration of vaccines globally; adjunct prevention and control measures are going to need to be continued in the months to come.

Patients with end stage kidney disease (ESKD) have been identified as having a poor prognosis following SARS-CoV-2 infection (6) (7) (8) . In addition, it is also recognised that patients receiving incentre haemodialysis (ICHD) are at higher risk of acquiring infection due to the inability to shield effectively (8) . Using serological methods, we have previously shown that ESKD patients readily seroconvert following confirmed SAR-CoV-2 infection; we have also shown that asymptomatic seroconversion is common in the high exposure setting of ICHD units (7) . What is not currently known in this population, is the durability of detectable immune responses and whether the presence of SARS-CoV-2 antibodies protects an individual with ESKD from reinfection.

In this study, we report the longitudinal serological status of a large cohort of ICHD patients. The aim of our study was to compare the longevity of the antibodies to the different SARS-CoV-2 antigenic targets, namely the nucleocapsid and receptor binding domain of the spike protein. We investigate cellular immune responses in patients in whom antibody responses have waned, and finally we evaluate whether immune responses to SARS-CoV-2 infection protect dialysis patients from subsequent reinfection.

Three-hundred and fifty-six patients receiving ICHD within 2 units affiliated with Imperial College Renal and Transplant Centre as previously reported, were included(7). Patients were followed up from 24 th February 2020 until 1 st January 2021. All patient samples (n=356) at time 0 were tested for Nprotein and RBD antibodies. At 6 months, all patient samples (n=301) were tested for N-protein antibodies; samples which were either anti-NP+ or indeterminant were tested for RBD antibodies. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 26, 2021.

Patient outcomes, including all new SARS-CoV-2 infections, confirmed by viral detection, were recorded up until the 1 st January; which incorporates data from the 2 nd wave of infections in the UK.

The study was approved by the Health Research Authority, Research Ethics Committee (Reference: 20/WA/0123 -The Impact of COVID-19 on Patients with Renal disease and Immunosuppressed Patients).

Baseline serum from all patients were tested for Nucleocapsid protein (NP) using the Abbott Architect SARS-CoV-2 IgG 2 step chemiluminescent immunoassay (CMIA) assay according to manufacturer's instructions. For this study, samples were interpreted as positive or negative according to the manufacturer's instructions with a cut off index value of 1.4 (9) . All baseline serum underwent testing for total receptor binding domain (RBD) antibodies using an in-house double binding antigen ELISA (Imperial Hybrid DABA; Imperial College London, London, UK), which detects total RBD antibodies (10) . The in house assay cut off was calculated from receiver operating characteristic curve analysis, and serum reactivity normalized by using the signal-to-cutoff ratio (S/CO); the ratio of optical density values generated in a sample to the cutoff optical density value. For this study, a sample was considered antibody positive if the S/CO was >1.2. At 6 months all samples were re-tested for NP antibodies. Samples with positive or equivocal NP antibodies (cut off-index (0.25-2.5)) were tested for RBD antibodies. overlapping SARS-CoV-2 structural peptide pools; S protein (spike protein), N protein (nucleocapsid protein), M protein (membrane), S1 and S2 spike protein components as well as positive and negative controls. Cells were incubated and interferon-γ secreting T cells were detected. The sum of T-SPOT immune responses to SARS-CoV-2 structural peptides was calculated. Counts more than 12 spots per 250,000 PBMC were reported as positive (11) .

Infection with SARS-CoV-2 was confirmed through reverse-transcriptase polymerase chain reaction (RT-PCR) assay of nasopharyngeal swab specimens, either following routine screening or acute presentation. Reverse-transcriptase PCR was carried out as per Public Health England (PHE) All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 26, 2021. ; guidelines, utilising certification marked assays with primers directed against multiple targets of SARS-CoV-2 genes (12). Routine screening of patients for the development of symptoms or a fever occurred prior to each haemodialysis session from the 9 th March onwards. In addition, routine asymptomatic swabbing was performed in June, and recommenced weekly during the second surge from 15 th November.

Statistical and graphical analyses were performed with MedCalc® v19.2.1. The two-sided level of significance was set at p<0.05. Chi-squared testing were used for proportional assessments. Nonparametric data were compared with the Mann-Whitney test. The Wilcoxon test was used to compare antibody levels of paired samples. Using the log rank test, Kaplan-Meier analyses were used to estimate and compare the risk of infection (or re-infection) by serological status. We recorded any positive PCR test at >60 days following a positive serological test at Time 0, to prevent capture of persistent viral detection of the primary infection(13). As we were not routinely PCR swabbing all asymptomatic cases at the time of first serological sampling, we also used only the PCR results taken >60 days post serological screening in the antibody negative group. Subsequent PCR positive free survival was censored for death, in the absence of PCR confirmation and transplantation.

At Time 0, 129/356 (36.2%) of all patients had detectable anti-NP and 134/356 (37.6%) had detectable anti-RBD. The clinical characteristics of patients by anti-NP status has been described previously and are shown in Table 1(7) .

Three hundred and one patients had a sample available at 6 months post initial sampling. Of 190 patients who were anti-NP-at Time 0, 6 (3.2%) had detectable anti-NP by month 6; 3 of which had PCR proven disease in the intervening period. In the patients who were anti-NP+ at time 0; the (S/C) was significantly higher in symptomatic patients compared with asymptomatic patients, with a median value of 7.3 (IQR: 6.1-8.5) and 6.2 (3.2-7.1) respectively, p=0.0006. 111/129 patients who were anti-NP+ at Time 0 had a sample available at 6 months. 40/111 (36.0%) were subsequently found to be anti-NP-at 6 months. In patients who had a detectable anti-NP antibody at both time 0 and 6 months; the All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

Twelve patients who anti-NP+ at Time 0, were seronegative for both anti-NP and anti-RBD at 6 months.

T-cell responses were investigated in 11 of these patients. One of these patients who was anti-RBD-at both Time 0 and 6 months, was also found to have unreactive antigen specific T-cell responses. Of the remaining 10 patients, 8 had positive ELISPOT readouts as shown in Table 2 . Both patients with a negative ELISPOT test were over the age of 70 years, one had a history of bladder cancer, but neither were iatrogenically immunosuppressed. T-cell responses were not available on patients who were anti-NP-but anti-RBD+ at Time 0.

Combining the results of the above immunological assessment, from the original 129 patients who were anti-NP positive, 126 (97.7%) had evidence of persistence of either serological or cellular immune responses against SARS-CoV-2 at 6 months. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Patients with anti-RBD were also at lower risk of being diagnosed with SARS-CoV-2 by PCR testing compared with anti-RBD-patients, p=0.0051 (log-rank), as shown in figure 4 . Of the 7 anti-RBD+ patients, who were anti-NP-at Time 0, 2 subsequently went on to have a PCR-positive test at >60 days; the first was diagnosed at day 74 post serological test, and this may represent persistent viral carriage rather than reinfection. The 2 nd case was a male in his 70s who was diagnosed at day 112 following a symptomatic infection who has subsequently made a full recovery.

We have shown that immune responses to natural SARS-CoV-2 infection in ICHD patients are durable for up to 6 months, even in patients who had mild or asymptomatic infection. Furthermore, we have provided data that shows that an immune response to SARS-CoV-2 infection may help provide protection against 'infection' or reinfection in dialysis patients in the medium term. Using serological status to determine previous exposure in ICHD populations, may therefore help identify patients at higher risk of primary infection whilst awaiting vaccine administration.

A recent large longitudinal study of healthcare workers has shown that the presence of anti-spike or anti-nucleocapsid antibodies was associated with a reduced risk of reinfection over a 6 month period(13, 14) . This data is consistent with the relative sparsity of reports of reinfection in the literature (15, 16) .

However, given this study included healthcare workers, who are likely to be significantly younger and lack co-morbidity, translating these findings to patients with ESKD in the absence of data would be injudicious. However, the findings of the healthcare worker study are supported by results from a separate report which investigated the medium term humoral and cellular responses in SARS-CoV-2 patients with a range of disease severity and co-morbidities, which showed that robust immune responses may persist for at least 8 months post-infection (17) . Whilst this may be more comparable data for dialysis patients, a further study from this same research group also showed a disparity in mounting adaptive immune responses in older persons(18). This is of concern for the nephrology community, as dialysis patients are also known to have impairment of both the innate and adaptive immune responses which correlate with premature ageing (19) .

Our data, which is the first to investigate longevity of SARS-CoV-2 immune responses in dialysis patients, is therefore of clinical importance. Like others, we have shown that antibody levels wane over time and rate of decay correlates with infection severity (20) . However, we acknowledge that use of seroprevalence alone may underestimate ongoing immunity to SARS-CoV-2; with data showing that robust T-cell responses maybe detectable in patients who have had mild or asymptomatic disease, even in the absence of antibodies (21) . In our dialysis cohort, we have shown that less than 3% of patients lacked evidence of either serological oor T-cell responses at 6 months post infection. Of upmost clinical relevance, we have also shown that a detectable serological response to SARS-CoV-2 infection, appears All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in to be protective against reinfection in dialysis patients, even at 6 months post-infection. This data is encouraging given the high rates of infection seen during the early stages of pandemic, as whilst dialysis patients await vaccination, previous exposure to infection may offer some protection. It is worth noting that of the 2 patients with serological evidence of anti-NP, who had subsequent viral detection by RT-PCR at days 142 and 205 post initial detection of seroconversion, one patient has HIV and the other patient had previously had a kidney transplant (Supplemental Table 1 ). In addition, the 2 patients with no serological or cellular evidence of immunity at 6 months, were both over the age of 70 years. Therefore, although we have shown that dialysis patients per se appear to mount a protective immunological response to SARS-CoV-2, there may be additional recognised clinical factors which may impair immunity and influence outcome in dialysis patients, which requires further study.

Whilst we have shown the development and sustainability of post-infection SARS-CoV-2 immune responses, we still do not know whether this high-risk patient population will mount an adequate vaccine response. To date, patients with ESKD, whether on dialysis or transplanted, have been excluded from the SARS-CoV-2 vaccine trials. Dialysis patients are recognised to have lower rates of seroconversion to vaccinations compared with healthy controls, which in part is due to the impact of uraemic toxins on the immune response (22) . This also translates into higher sepsis related mortality rates in dialysis patients compared with the general population (23) . This immune deficiency appears to be related to responses to new antigenic pathogens, which of vital importance in the outcome to SARS-CoV-2 vaccination (24) . As there is no ESKD efficacy data available from any of the SARS-CoV-2 vaccines to date, we can only hypothesise from previous vaccination programmes that seroconversion in dialysis patients will be inferior than the general public. Extrapolating from the knowledge that the immunosenescence associated with aging is seen prematurely in dialysis patients, and that older people had significantly lower levels of anti-spike protein antibody and neutralising antibody levels at 28 days post the BNT162b2 injection than younger patients; suggests the urgent need for some prospective data in this vulnerable population (5, 25) . Furthermore, baseline serological status of patients from past exposure may be of relevance in ESKD patients, and it will be of interest to evaluate whether vaccine responses are inferior in infection naïve patients (26) .

This study has several limitations, in part due to the time sensitive nature of the results which have led us to take a pragmatic approach to sample processing. The study would have been strengthened by the addition of more laboratory data on viral loads over time. Certainly, there have been case reports of prolonged viral shedding, which may be more common in immunosuppressed patients (27, 28) .

However, we used a 60-day cut off for 'new' PCR positivity, as utilised by others previously(13). It is also relevant to highlight that all patients did subsequently undergo routine asymptomatic PCR swabbing after this time point. A further limitation is that we do not have data available on viral genetic sequencing of infected patients, and it is possible that 'reinfections' are due to new variants of SARS-CoV-2, which may evade immune responses to previous strains (29) . However, if this was the case, we All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in shown to more closely correlate with neutralising antibody titre (30) . Finally, we only performed T-cell ELISPOT assays in selected cases as time and resource limitations prevented further testing at this stage. However, the major strengths of our study, is that to our knowledge this is the first report of longitudinal immunological responses in dialysis patients, which in addition has been correlated with clinical outcome data in asymptomatic and symptomatic infections. perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted January 26, 2021. ; perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.22.21249865 doi: medRxiv preprint b. Receptor Binding Domain Antibody 97/111 (87.4%) of patients with anti-RBD at Time 0 became retained their antibodies at 6 months. Of which, the anti-RBD index (S/C)) was significantly higher at Time 0 compared with 6 months post testing, with a median (S/CO) of 23.9 (IQR: 23.4-26.1) and 23.4 (IQR: 8.9-24.1) respectively, p=0.0004.. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

From >60 days after initial serological testing, anti-NP+ patients were at significantly lower risk of being diagnosed with SARS-CoV-2 compared with anti-NP-patients, p=0.0005 (logrank)

All rights reserved. No reuse allowed without permission.

perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.22.21249865 doi: medRxiv preprint

From >60 days after initial serological testing, anti-RBD+ patients were also at lower risk of being diagnosed with SARS-CoV-2 by PCR testing compared with anti-RBD-patients, p=0.0051 (log-rank) All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.22.21249865 doi: medRxiv preprint

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