key: cord-0736713-az643ps0
authors: Clarke, Candice L.; Prendecki, Maria; Dhutia, Amrita; Gan, Jaslyn; Edwards, Claire; Prout, Virginia; Lightstone, Liz; Parker, Eleanor; Marchesin, Federica; Griffith, Megan; Charif, Rawya; Pickard, Graham; Cox, Alison; McClure, Myra; Tedder, Richard; Randell, Paul; Greathead, Louise; Guckian, Mary; McAdoo, Stephen P.; Kelleher, Peter; Willicombe, Michelle
title: Longevity of SARS-CoV-2 immune responses in hemodialysis patients and protection against reinfection
date: 2021-03-25
journal: Kidney Int
DOI: 10.1016/j.kint.2021.03.009
sha: 57a4925bc918a0817177a99108949637fc6b514e
doc_id: 736713
cord_uid: az643ps0

Patients with end stage kidney disease receiving in-center hemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, patients receiving ICHD frequently develop circulating antibodies to SARS-CoV-2, even with asymptomatic infection. Here, we investigated the durability and functionality of the immune responses to SARS-CoV-2 infection in patients receiving ICHD. Three hundred and fifty-six such patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were regularly screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies, and those who became seronegative at six months were screened for SARS-CoV-2 specific T-cell responses. One hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at time zero, of whom 127 also had detectable anti-RBD. Significantly, at six months, 71/111 (64.0%) and 99/116 (85.3%) remained anti-NP and anti-RBD seropositive, respectively. For patients who retained antibody, both anti-NP and anti-RBD levels were reduced significantly after six months. Eleven patients who were anti-NP seropositive at time zero, had no detectable antibody at six months; of whom eight were found to have SARS-CoV-2 antigen specific T cell responses. Independent of antibody status at six months, patients with baseline positive SARS-CoV-2 serology were significantly less likely to have PCR confirmed infection over the following six months. Thus, patients receiving ICHD mount durable immune responses six months post SARS-CoV-2 infection, with fewer than 3% of patients showing no evidence of humoral or cellular immunity.

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 in-J o u r n a l P r e -p r o o f centre 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 nucleocapsid protein (anti-NP) and RBD (anti-RBD) antibodies. At 6 months all available samples (n=301) were re-tested for anti-NP (Supplemental Data Figure 1 ). In addition, samples were tested for anti-RBD if any of the following criteria were met; patients were anti-NP+ at 6 months, patients had an equivocal anti-NP result (i.e a cutoff index (S/C) of 0.25-1.39) or were anti-NP-(i.e. a S/C of ≤0.24) at 6 months but were either anti-NP and/or anti-RBD positive at baseline.

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.

A diagramatic overview of the outcome of patients by serological and symptomatic status is shown in 

Baseline serum from all patients were tested for both nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies. The presence of anti-NP was assessed using the J o u r n a l P r e -p r o o f commercially available 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 cutoff index value of 1.4 (9) .

Anti-RBD was detected 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). For this study, a sample was considered antibody positive if the S/CO was >1.2. An RBD assay was used in addition to an anti-NP assay, as anti-RBD has been shown to correlate with anti-neutralising antibodies (11) . spots per 250,000 PBMC were reported as positive (12) .

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) guidelines, utilising certification marked assays with primers directed against multiple targets of SARS-CoV-2 genes (13). Between March and June 2020, patients underwent RT-PCR testing of nasopharyngeal swabs when they presented for dialysis with symptoms. In June, all patients in our centre were screened regardless of symptoms, as part of a single surveillance exercise to ascertain prevalent infection. From the start of the second wave in November 2020, all ICHD patients underwent weekly routine RT-PCR testing of nasopharyngeal swabs.

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. Non-J o u r n a l P r e -p r o o f parametric 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(14). 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. Table 1(7) . 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 protein antibodies was associated with a reduced risk of reinfection over a 6 month period(14, 15). These data are consistent with the relative sparsity of reports of reinfection in the literature (16, 17) . 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, a separate report, which investigated the medium term humoral and cellular responses in SARS-CoV-2 patients with a range of co-morbidities, has also shown that robust immune responses may persist for at least 8 months post-infection (18) . Whilst this may be more comparable data for dialysis patients, a further study from this same research group showed there was a disparity in adaptive immune responses in older persons(19). 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 (20) .

Our study, 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 (21) . We found that symptomatic infection is associated with higher antibody 'titres', and it was reassuring to demonstrate the distribution of S/C values in dialysis patients post-infection is comparable with healthcare workers as reported in other studies using the same serological assay (22) . Furthermore, consistent with data in healthcare workers, we have also shown that an anti-RBD immune response is more durable than an anti-NP response (22) . However, we acknowledge that use of seroprevalence alone may underestimate ongoing immunity to SARS-CoV-2; with data showing that robust T-cell responses can be detected in patients who have had mild or asymptomatic disease, even in the absence of antibodies (23) . In our dialysis cohort, we have shown that fewer than 3% of patients lacked evidence Table 1 ). In addition, the 3 patients with no serological or cellular evidence of immunity at 6 months, all were over the age of 70 years. Therefore, although we have shown that dialysis patients per se appear to mount an 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.

The efficacy of SARS-CoV-2 vaccines in patients with ESKD is currently not known as such patients were excluded from the preliminary SARS-CoV-2 vaccine trials. Dialysis patients are recognised to have lower rates of seroprotection to vaccinations compared with healthy controls, which in part is due to the impact of uraemic toxins on the immune response (24) . This immune deficiency appears to be related to responses to new antigenic pathogens, which is of vital importance in the outcome to SARS-CoV-2 vaccination (25) . 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, 26) . Reasurringly data are now emerging that neutralising antibodies developed in response to SARS-CoV-2 vaccination are more durable than immunity from natural infection (27) . This coupled with the data we present of sustained serological responses and protection from reinfection akin to those in health care workers, provides hope for comparable vaccine responses in patients with ESKD(14, 27). Furthermore, baseline serological status of patients from past exposure may be of relevance in ESKD patients given the high prevalence of infection in patients receiving ICHD, and it will be of interest to evaluate whether vaccine responses are less robust in infection naïve patients (28) .

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 (29, 30 Figure S1 . Schematic diagram of criteria to test samples by assay type at Time 0 (T0) and at Time 6 (T6) months Table S1 . Characteristics of 5 seropositive patients who were subsequently found to be SARS-CoV-2 positive 

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)

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) 

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