Humoral Responses in the Omicron Era Following 3-Dose SARS-CoV-2 Vaccine Series in Kidney Transplant Recipients : Transplantation Direct

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Kidney Transplantation

Humoral Responses in the Omicron Era Following 3-Dose SARS-CoV-2 Vaccine Series in Kidney Transplant Recipients

McEvoy, Caitríona M. MB, BCh, PhD1,2,3; Hu, Queenie PhD4; Abe, Kento T. BSc4,5; Yau, Kevin MD3,6; Oliver, Matthew J. MD, MHS3,6,7; Levin, Adeera MD8; Gingras, Anne-Claude PhD4,5; Hladunewich, Michelle A. MD, MS3,6,7; Yuen, Darren A. MD, PhD1,2,3

Author Information
Transplantation Direct 9(1):p e1401, January 2023. | DOI: 10.1097/TXD.0000000000001401

Abstract

INTRODUCTION

Solid organ transplant recipients (SOTRs) and patients with chronic kidney disease are at increased risk for SARS-CoV-2 infection and adverse outcomes.1–4 Consequently, kidney transplant recipients (KTRs) represent an especially vulnerable population and a priority group for vaccination. Although offering some protection, the humoral response observed following 2 doses of SARS-CoV-2 vaccine among SOTRs in general, and KTRs in particular, is inferior to that of immunocompetent individuals, as is the real world effectiveness of 2-dose vaccination.5–11

The improved immunogenicity observed in SOTRs following a third dose of mRNA vaccine12–15 led to the recommendation for a primary vaccine series of 3 doses in this population.16 Studies have shown augmented binding antibody levels and enhanced neutralizing capabilities following the third vaccine dose in SOTR cohorts, at 1 and 3 mo timepoints.17,18 However, diminished responses in KTRs relative to other organ groups are reported,5,19 and data on the durability of the antibody response beyond 1 mo in KTR cohorts are lacking.

In late 2021, the Omicron SARS-CoV-2 variant emerged and rapidly gained dominance worldwide. The first Omicron wave related to the BA.1 (B.1.1.529.1) subvariant; however, in recent months, successive Omicron subvariants (BA.2-BA.5) have predominated.20–22 Because of a large number of mutations in the receptor binding domain (RBD)—the main target of neutralizing antibodies—Omicron subvariants can substantially evade the pre-existing humoral neutralization response induced by vaccines or infection.23–27 Importantly, in comparison to a 2-dose strategy, a third dose of an mRNA SARS-CoV-2 vaccine has been shown to substantially boost neutralizing antibody titers against Omicron in the general population26,28 and to reduce overall infection rates and mortality.29,30 However, data on neutralizing capacity of KTR plasma to Omicron after additional vaccine doses remain limited. Published studies to-date have focused only on SOTRs31 or have short (1 mo) follow-up.32,33

A deeper understanding of the immune response that is predictive of protection from Omicron subvariants would direct future vaccine and pre-exposure prophylaxis strategies in this vulnerable population. Therefore, we conducted a prospective observational study in a cohort of KTRs to comprehensively profile the binding and neutralizing antibody responses at 1 and 3 mo following the third vaccine dose and to compare the induced neutralization response with a cohort of healthy controls.

Our study is an important addition to the existing literature related to SARS-CoV-2 vaccine-induced responses in KTRs. In particular, it is the first to provide longitudinal data (with follow-up of 3 mo, compared with similar studies, which have limited their immunogenicity timepoint to 1 mo postvaccination) and more importantly, to carefully profile multiple parameters of humoral immunity (including type, concentration, and seropositivity rate), and their association with Omicron neutralization using a potentially scalable antibody platform. These data add to the accumulating evidence on potential correlates of protection that clinicians and policymakers can use to guide decision-making for this patient population.

MATERIALS AND METHODS

Patient Population and Study Design

We conducted a prospective observational cohort study of KTRs followed by the Renal Transplant Program of St. Michael’s Hospital (Unity Health Toronto), Toronto, Ontario. Adult patients (≥18 y) who had received a kidney transplant and had a functioning allograft were considered eligible for inclusion. Prevalent and incident patients were invited to participate in September 2021 when eligibility for third vaccine dosing in SOTRs was confirmed by the Ontario Ministry of Health.

Clinical Data Sources

Patient demographics, clinical characteristics and covariates, and SARS-CoV-2-related data regarding vaccination or infection status were collected from St. Michael’s Hospital electronic health records.

Healthy Controls

Healthy controls included healthcare workers and research staff recruited at Sunnybrook Health Sciences Centre. Healthy controls were ≥18 y old and in self-reported good health. Individuals with chronic conditions including chronic kidney disease, diabetes mellitus, hypertension, HIV, usage of immunosuppressants, and prior organ transplantation were excluded.

Serologic Assays

Anti-spike, anti-RBD‚ and anti-nucleocapsid SARS-CoV-2 IgG antibodies were measured using an automated enzyme-linked immunosorbent assay (ELISA) as previously described.34,35 Antibody levels were reported as relative ratios to a synthetic standard included as a calibration curve on each assay plate. Our ELISA-based assay was harmonized with the WHO international standard unit—the Binding Antibody Unit. A table for conversion from relative ratios to Binding Antibody Unit/mL is provided in Table S1 (SDC, https://links.lww.com/TXD/A470).

Thresholds for seropositivity (seroconversion) were determined as ≥3 standard deviations from the log means of aggregated data from 300 archived (pre-SARS-CoV-2-era) negative sera.34 Seroconversion/seropositivity thresholds were 0.19, 0.186‚ and 0.396 for anti-spike, anti-RBD, and anti-nucleocapsid antibodies, respectively. We considered the median levels of convalescent serum (taken 21–115 d after symptom onset in a cohort of 211 patients in the general population with a median age of 59 y who had mild to severe SARS-CoV-2), as reflective of a robust antibody response34,35; the medians were 1.38, 1.25, and 1.13 for anti-spike, anti-RBD, and anti-nucleocapsid antibodies, respectively. Expanded details are provided in Supplemental Methods.

Spike-pseudotyped Lentivirus Neutralization Assay

The neutralization assay was adapted from a previously validated SARS-CoV-2 spike-pseudotyped lentivirus assay with constructs for Beta, Delta‚ and Omicron variants, with minor modifications.36 Full details are provided in Supplemental Methods.

Statistical Analysis

Demographic and clinical characteristics were compared using Wilcoxon rank sum test for continuous variables and Fisher’s exact test for categorical variables. For paired samples, differences in anti-RBD and anti-spike antibody levels were compared using the Wilcoxon signed-ranks test; differences in proportions were assessed using McNemar’s test with continuity correction. Receiver operating characteristic (ROC) curve analysis was performed using the pROC package.37 To maximize specificity, the optimal decision threshold was identified using the “closest.topleft” metric. Antibody titer calculations were performed in GraphPad Prism 9 (GraphPad Software, San Diego, USA), all subsequent analyses were performed in R, version 4.1.2.38

2. 7. Study Approval

This study was registered with Clinical Trials Ontario and was approved by the Research Ethics Boards of Sunnybrook Health Sciences Centre Research Ethics Board and St. Michael’s Hospital (Project ID 3604). All participants provided written informed consent before study participation.

RESULTS

Patient Characteristics and Baseline Humoral Response

Pre- and post-third-dose blood samples were available for 44 KTRs. Baseline demographic and clinical characteristics are shown in Table 1. Median age was 55.5 y (interquartile range [IQR], 45.8–63 y), and the majority (79.5%) of the population was male. The median time from transplant was 43.1 mo (IQR, 7–142 mo). All patients were on a calcineurin inhibitor, with 91% taking tacrolimus. Seventy percent were taking an antimetabolite (mycophenolic acid-based in all cases), 93% of patients were taking prednisolone, and in total, 31 of 44 (70.5%) were on a standard triple-agent immunosuppressive regimen at the time of the third vaccine dose. All participants had previously received 2 doses of an mRNA-based vaccine, with 35 (79.5%) having received Pfizer-BioNTech BNT162b2, and 8 (18.1%) having received Moderna mRNA-1273. Twenty-six of 44 patients (59%) were also tested at 3 mo post-third dose. This group had similar clinical and demographic characteristics to the parent cohort (Table 1).

TABLE 1. - Characteristics of the study cohort
Pre–third dose and month 1 blood samplesN = 44 Pre–third dose, month 1‚ and month 3 blood samplesN = 26
Male sex, N (%) 35 (79.5) 23 (88.5)
Age (y) at dose 3Median (IQR) 55.5(45.8–63) 56(48.8–63.8)
Transplant vintage at dose 3 (mo)Median (IQR) 43.1(7–142) 39.6(10.2–145.4)
eGFR (mL/min/1.73 m2)Median (IQR) 53(36.3–73.6) 48.9(35.7–64.9)
Donor type, N (%)
 Deceased donor 29 (66) 18 (69.2)
 Living donor 15 (34) 8 (30.8)
Immunosuppression at time of dose 3, N (%)
 Prednis1 41 (93.2) 24 (92.3)
 Antimetabolite (mycophenolate sodium/mofetil) 34 (77.3) 20 (77)
 CNI—tacrolimus 40 (91) 23 (88.5)
 CNI—cyclosporine 4 (9) 3 (11.5)
 On triple-agent immunosuppression 31 (70.5) 18 (69.2)
BMI kg/m2Median (IQR) 25.7(23.4–29.5) 26.5(23.9–29.5)
Previous COVID-19 infection, N (%) 2 (4.5) 1 (3.8)
Initial (2 dose) vaccine series, N (%)
 Pfizer-BioNTech (BNT162b2) 35 (79.5) 22 (84.6)
 Moderna (mRNA-1273) 8 (18.1) 3 (11.5)
 Mixed 1 (2.3) 1 (3.8)
3-dose vaccine series, N (%)
 Pfizer-BioNTech (BNT162b2) 35 (79.5) 22 (84.6)
 Moderna (mRNA-1273) 7 (15.9) 3 (11.5)
 Mixed 2 (4.6) 1 (3.8)
Dose and blood testing intervals (d)Median (IQR)
 Dose 1 to dose 2 21(21–31) 21(21–27.5)
 Dose 2 to dose 3 152(127.3–185.7) 134.5(122–158.5)
 Dose 2 to prebooster blood testing 149.5(124–174) 133.5(119.5–154.5)
 Dose 3 to month 1 blood testing 26.5(20.75–32.25) 25.5(18.8–29.8)
 Dose 3 to month 3 blood testing NA 84.5 (79.5–90)
BMI, body mass index; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate; IQR, interquartile range.

The baseline demographics of the healthy controls (n = 13) are shown in Table S2 (SDC, https://links.lww.com/TXD/A470). Their median age was 46 y (IQR, 31–55 y), and 30.7% of the population was male. All healthy controls had received the Pfizer-BioNTech BNT162b2 vaccine for their initial 2 doses.

Anti-RBD, anti-spike, and anti-nucleocapsid IgG were measured in plasma before and after a third dose of an mRNA-based SARS-CoV-2 vaccine in KTRs. At a median time of 149.5 d (IQR 124–174) postsecond vaccine dose, 24 of 44 (54.5%) and 33 of 44 (75%) KTRs were seropositive for anti-RBD and anti-spike antibodies, respectively (Table 2). However, just 6.8% (3 of 44) and 18.2% (8 of 44) of patients had anti-RBD and anti-spike antibody levels consistent with a robust antibody response (ie‚ exceeding the median convalescent serum levels seen in healthy controls34)(Figure 1A and B). Before the third dose, previous SARS-CoV-2 infection had been confirmed by PCR- or rapid antigen-testing in 2 of 44 patients. However, only 1 of the cases with prior SARS-CoV-2 infection was seropositive for anti-nucleocapsid antibody (Figure S1A, SDC, https://links.lww.com/TXD/A470). An additional patient with no known history of SARS-CoV-2 infection was also seropositive for anti-nucleocapsid antibody. In both cases, the titers remained below the median convalescent response of healthy controls.34

TABLE 2. - Summary of binding and neutralizing antibody profiles of KTRs before, and at 1 and 3 mo after third mRNA SARS-CoV-2 vaccine dose
Binding antibody Relative ratio (median [IQR]) No. (%) participants with seropositivity No. (%) participants with antibody levels ≥ median convalescent level
Prethird doseN = 44 1 mo post-third doseN = 44 3 mo post-third doseN = 26 Pre–third doseN = 44 1 mo post-third doseN = 44 3 mo post-third doseN = 26 Pre–third doseN = 44 1 mo post-third doseN = 44 3 mo post-third doseN = 26
Anti-spike 0.871(0.218–1.264) 1.447(1.149–1.549) 1.454(0.535–1.60) 33 (75) 39 (88.6) 23 (88.4) 8 (18.2) 28 (63.6) 14 (53.8)
Anti-RBD 0.212(0.033–0.597) 1.198(0.249–1.478) 0.479(0.066–1.373) 24 (54.5) 33 (75) 15 (57.6) 3 (6.8) 20 (45.5) 8 (30.7)
Anti-nucleocapsid 0.063(0.044–0.097) 0.06(0.046–0.084) 0.0597(0.0489–0.069) 2 (4.5) 2 (4.5) 0 (0) 0 (0) 0 (0) 0 (0)
Neutralizing antibody Log 10 ID 50 (median [IQR]) (of responding patients) No. (%) participants with detectable neutralizing antibody
Prethird doseN = 44 1 mo post-third doseN = 44 3 mo post-third doseN = 26 Pre–third doseN = 44 1 mo post-third doseN = 44 3 mo post-third doseN = 26
Wild type 2.20 (1.81–2.56) 3.28 (2.62–3.55) 2.84 (2.18–3.40) 21 (47.7) 32 (72.2) 16 (61.5)
Beta 1.68 (1.53–1.99) 2.76 (2.26–3.15) 2.51 (2.19–3.08) 11 (25) 25 (56.8) 12 (46.2)
Delta 2.06 (1.74–2.34) 3.01 (2.52–3.37) 2.66 (2.09–3.21) 14 (31.8) 28 (63.6) 14 (53.9)
Omicron 0 (0–0) 2.49 (1.87–2.87) 2.16 (1.87–2.66) 0 (0) 20 (45.5) 10 (38.5)
IQR, interquartile range; RBD, receptor-binding domain.

F1
FIGURE 1.:
Binding antibody response at 1 and 3 mo post-third mRNA-vaccine dose. Levels of (A) serum anti-RBD and (B) anti-spike IgG in 44 participants with blood samples drawn pre- and at 1 mo post-third dose. Anti-RBD and anti-spike antibody levels had significantly increased in the study population at 1 mo post-third dose (Wilcoxon signed rank test, P  = 3.65 × 10–10 and 7.51 × 10–10, respectively). The proportion of kidney transplant recipients now exhibiting a robust antibody response with both anti-spike antibody (McNemar test with continuity correction, P = 5.1 × 10–5) and anti-RBD antibody (McNemar test with continuity correction, P = 0.001) was significantly increased as shown. Levels of (C) serum anti-RBD and (D) anti-spike IgG in 26 participants with additional blood samples drawn at 3 mo post-third dose. Anti-RBD and anti-spike antibody levels had decreased at 3 mo post-third dose (Wilcoxon signed rank test, P = 8.2 × 10–5 [anti-RBD] and P = 0.84 [antispike]). The proportion of kidney transplant recipients now exhibiting a robust antibody response with both anti-spike antibody (McNemar test with continuity correction, P = 1) and anti-RBD antibody (McNemar test with continuity correction, P = 0.48) was not significantly altered as shown. For all images: the values depicted are relative ratios against a synthetic standard. Serum volume 0.0625 μL. Threshold lines and marked values demonstrate seropositivity (green-dashed line) and the median convalescent response (blue-dashed line). Individual values are colored to depict the response level as shown in the legend. Solid black lines indicate the median ratio values for each grouping. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. RBD, receptor binding domain.

Humoral Response at 1 and 3 Mo Post-third mRNA-vaccine Dose

At a median of 26.5 d post-third vaccine dose, 33 (75%) and 39 (88.6%) KTRs were seropositive for anti-RBD and anti-spike antibodies, respectively. In comparison to pre–third dose serology, anti-spike and anti-RBD antibody levels had significantly increased in our KTR cohort (anti-spike fold-increase 1.66, anti-RBD fold-increase 5.65; P = 3.65 × 10–10 and 7.51 × 10–10, respectively) (Figure 1A and B).

A greater proportion of KTRs now exhibited a robust response, as defined by anti-spike antibody (28 of 44 [63.6%] participants, P = 5.1 × 10–5) and anti-RBD antibody (20 of 44 (45.5%), P = 0.001) values above the median levels measured in convalescent healthy controls (Figure 1A and B). Although a substantial number of participants remained seronegative at all timepoints tested, 6 of 11 (54%) and 9 of 20 (45%) of patients who were seronegative at baseline had seroconverted with respect to anti-spike and anti-RBD antibodies when assessed 1 mo post-third vaccine dose. Anti-spike and anti-RBD antibody levels were highly correlated among individuals (Spearman’s rho 0.82, P < 2.2 × 10–16, Figure S1B, SDC, https://links.lww.com/TXD/A470).

In total, 35 of 44 (79.6%) and 7 of 44 (15.9%) of KTRs received 3 doses of BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna), respectively, with 2 KTRs (4.5%) receiving a mixed-vaccine series. The majority of those who received mixed-vaccine, or mRNA-1273-only regimens had binding antibody titers consistent with a robust response at month 1; however, differences in proportions were not significant (although anti-RBD overall P = 0.001, post hoc adjusted P values were 0.09 [all BNT162b2 versus all mRNA-1273], 0.14 [all BNT162b2 versus mixed], and 1 [all mRNA-1273 versus mixed]; antispike overall P = 0.59) (Figure S2A,B, SDC, https://links.lww.com/TXD/A470). Likewise, median anti-RBD and anti-spike antibody levels were not significantly different between BNT162b2-only, mRNA-1273-only‚ and mixed-vaccination groups (anti-RBD P = 0.1, anti-spike P = 0.22, Figure S2C,D, SDC, https://links.lww.com/TXD/A470).

In 26 of 44 patients, antibodies were further assessed at 3 mo. At a median time of 84.5 d post-third vaccine dose, 15 (57.7%) and 23 (88.5%) of patients were seropositive for anti-RBD and anti-spike antibodies, respectively. There was a significant decrease in the anti-RBD antibody levels of the overall group (P = 8.2 × 10–5), whereas the decline in anti-spike antibody levels was not significant (P = 0.87) (Figure 1C and D). Despite the decline in antibody levels, the proportion of patients exhibiting a robust antibody response with either anti-spike or anti-RBD was not significantly altered (anti-spike: P = 1, anti-RBD: P = 0.48). Exploring this further, we confirmed that participants who mounted a robust antibody response with either anti-spike or anti-RBD antibodies when tested 1 mo post-third dose, experienced a more modest attrition of antibody levels by month 3 when compared with patients who exhibited a partial response at month 1, as defined by seropositivity not reaching median convalescent levels in healthy controls (anti-RBD: P = 0.012, anti-spike: P = 0.57) (Figure S3A–C, SDC, https://links.lww.com/TXD/A470). Similar to the month 1 response, anti-spike and anti-RBD antibody levels remained highly correlated (Spearman’s rho 0.89, P = 1.6 × 10–6, Figure S4A, SDC, https://links.lww.com/TXD/A470). Finally, although 1 patient in this group had a prior history of SARS-CoV-2 infection, all patients were seronegative for anti-nucleocapsid antibody at this timepoint (Figure S4B, SDC, https://links.lww.com/TXD/A470).

F2
FIGURE 2.:
Comparison of neutralizing antibody levels in healthy controls and responding kidney transplant recipients pre- and post-third vaccine dose. For each variant, neutralizing antibody levels for responding KTRs, that is, those with detectable neutralizing antibody (Log10ID50 >0) were plotted alongside HCs and differences between medians was assessed using Wilcoxon rank sum test. * P ≤ 0.05, **P ≤ 0.01. HC, healthy control; KTR, kidney transplant recipient.

SARS-CoV-2 Neutralization at 1 and 3 Mo Following Third Dose

Neutralization capacity at baseline, and at 1 and 3 mo post-third vaccine dose was assessed for wild type (WT), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron BA.1) SARS-CoV-2 variants (Table 2). Before the third dose, 21 of 44 (47.7%), 11 of 44 (25%), and 14 of 44 (31.8%) participants had detectable neutralizing antibodies to WT, Beta, and Delta, respectively (Figure 2A). One month post-third dose, the majority of the cohort had detectable neutralizing antibody responses against WT (32 of 44 [72.7%]), Beta (25 of 44 [56.8%]), and Delta (28 of 44 [63.6%]) with median log10ID50 against WT: 3.28 (IQR, 2.62–3.55); Beta 2.76 (IQR, 2.26–3.15); and Delta 3.01 (IQR, 2.52–3.37) in the subgroup of positive patients. The proportion of participants with neutralizing antibodies against the WT, Beta, and Delta variants had significantly increased compared with baseline (WT: P = 0.026; Beta: P = 0.005; Delta P = 0.005) (Figure 2A). No participants had detectable neutralizing antibodies to Omicron prior to third-dose vaccination; however, this was detected in 20 of 44 (45.5%) of participants after 1 mo (P = 0.0037), with a median log10ID50 of 2.49 (IQR, 1.93–2.86) in the subgroup of positive patients. The proportion of participants with neutralizing antibody against Omicron was significantly lower than those with detectable neutralizing antibody against WT and delta variants, but not against Beta (P = 0.002 WT versus Omicron, P = 0.074 Beta versus Omicron, P = 0.013 Delta versus Omicron). We assessed the impact of vaccine type on the presence or absence of neutralizing antibodies against each variant; however, no significant associations were identified (WT: P = 0.81, Beta: P = 0.15, Delta P = 0.25, Omicron P = 0.21, Figure S5, SDC, https://links.lww.com/TXD/A470).

F3
FIGURE 3.:
Detection of neutralizing antibodies against SARS-CoV-2 wild type, Beta, Delta, and Omicron (BA.1) variants at 1 and 3 mo post-third mRNA-vaccine dose. (A) Neutralizing antibodies detected in 44 participants with blood samples drawn pre- and at 1 mo post-third dose. The proportion of kidney transplant recipients with detectable neutralizing antibody (Log10ID50 >0) was significantly increased for all variants (McNemar test with continuity correction; wild type P = 0.026, Beta P = 0.005, Delta P = 0.005; McNemar’s exact test: Omicron P = 0.0037). (B) Neutralizing antibodies detected in 26 participants with blood samples drawn pre- and at 1 and 3 mo post-third dose. The proportion of kidney transplant recipients with detectable neutralizing antibody (Log10ID50 >0) was not significantly altered compared with month 1 (McNemar test with continuity correction); P = 1 for all comparisons. For all images: paired values are linked with black dashed lines. Solid black lines in each violin plot indicate the median Log10ID50 values for each variant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

In the subgroup of patients with blood samples at 3 mo post-third dose, levels of neutralizing antibodies were reduced in comparison to the month 1 timepoint with median log10ID50 against WT: 2.84 (IQR, 2.18–3.40), Beta 2.51 (IQR, 2.19–3.08), Delta 2.66 (IQR, 2.09–3.21), and Omicron 2.16 (IQR, 1.87–2.66) in the subgroup of positive patients. Despite this decline, the proportion of patients with detectable neutralization response to individual variants had not significantly altered compared with the month 1 timepoint (P = 1 for all comparisons) (Figure 2B). At this time point, a smaller proportion of patients had detectable neutralizing antibodies against Omicron (10 of 26) as compared with WT (16 of 26, P = 0.041), Beta (12 of 26, P = 0.48), and Delta (14 of 26, P = 0.11).

As compared with WT, neutralizing antibody responses (log10ID50) were several fold-lower for Beta (median fold reduction 3.4 [IQR, 1–7.34]), Delta (median fold reduction 1.82 [IQR, 1–3.50]), and most strikingly, Omicron (median fold reduction 11.51 [IQR, 1–33.97]) in patients with detectable neutralizing antibodies at 1 mo and to a lesser extent at 3 mo (median fold reductions Beta [2.52 (IQR, 1–4.36)], Delta [1.55 (IQR, 1–2.62)], and Omicron [6.77 (IQR, 1–14.22)]; Figure S6, SDC, https://links.lww.com/TXD/A470).

Comparison of SARS-CoV-2 Antibody Responses With Healthy Controls

The presence and magnitude of binding and neutralizing antibody responses at 1 mo following the third vaccine dose in KTRs was next compared with a cohort of 13 healthy controls. The baseline demographics and vaccination details of this cohort are presented in Table S2 (SDC, https://links.lww.com/TXD/A470). The magnitude of binding antibody response among healthy controls was increased in comparison to KTRs (anti-spike 1.28 fold-increase, anti-RBD 1.58 fold-increase). In notable contrast to the KTRs, all healthy controls were categorized as exhibiting a robust response 1 mo post-third vaccine dose (Figure S7 and Table S3, SDC, https://links.lww.com/TXD/A470).

In contrast to KTRs, neutralizing antibodies against Omicron were detected in 5 of 13 (38.5%) healthy controls before receipt of the third vaccine dose. Additionally, 100% of healthy controls had detectable neutralizing antibodies against all variants, including Omicron, when tested at the 1 mo timepoint (Figure 3; Table S3, SDC, https://links.lww.com/TXD/A470). As observed in the transplant population, neutralization antibody responses were several fold-lower for Beta (median fold reduction 2.26 [IQR, 1.62–2.74]), Delta (median fold reduction 1.83 [IQR, 1.45–2.27]), and Omicron (median fold reduction 15.18 [IQR, 9.29–18.68]), versus WT at 1 mo in healthy controls (Figure S8, SDC, https://links.lww.com/TXD/A470).

F4
FIGURE 4.:
Comparison of neutralizing antibody levels detected in healthy controls and kidney transplant recipients pre- and post-third vaccine dose. Neutralizing antibodies detected in 44 KTRs and 13 healthy controls (HCs) before the third vaccine dose (prethird dose) and at month 1 post-third dose. For all images: Paired values are linked with black dashed lines. Solid black lines in each violin plot indicate the median Log10ID50 values for each variant. HC, healthy control; KTR, kidney transplant recipient.

We next focused on the subgroup of “responding KTRs,” that is, those who had detectable neutralizing antibody to an individual variant at 1 mo following the third vaccine dose. We compared the median log10ID50 of healthy controls with that of the responding KTRs and found that although the magnitude of response in relation to WT, Beta, and Delta variants was significantly lower in KTRs (median log10ID50 fold reduction in KTRs: WT [0.494, P = 0.003], Beta [0.641, P = 0.003], Delta [0.595, P = 0.002]), there was no significant difference in the median log10 ID50 of responding KTRs and our population of healthy controls against the Omicron variant (log10ID50 fold reduction in KTRs: Omicron [0.1, P = 0.27]) (Figure 4).

Assessment of Responders and Nonresponders

A number of individuals remained below the threshold of seropositivity for anti-spike and anti-RBD antibodies, or had no detectable SARS-CoV-2 neutralization capabilities. As Omicron subvariants currently dominate worldwide, we reasoned that one of the most important measurable humoral responses would be the presence of Omicron-specific neutralization antibodies. Therefore, we next explored if any demographic and transplant-related clinical factors of our study population associated with the development of Omicron-specific neutralizing antibodies. Individuals lacking a detectable Omicron-neutralizing response tended to be older, with a longer time posttransplant, a lower estimated glomerular filtration rate (eGFR), and to have received mainly deceased donor organs; however, no significant associations were identified (Table 3). Likewise, no baseline demographic or clinical/transplant characteristics identified people who were non-responders against all variants (n = 12), as compared with people with detectable neutralizing antibody against at least 1 variant (n = 32), though responders tended to have a longer interval time between receipt of vaccine doses 2 and 3 (Table 4). Serum levels of anti-spike and anti-RBD antibodies were significantly higher in Omicron-specific responders (compared with Omicron non responders) (anti-spike P = 1.1 × 10–7, anti-RBD P = 4 × 10–7) and in individuals who had detectable neutralizing antibody against 1 variant (compared with nonresponders for all variants) (anti-spike P = 1.1 × 10–7, anti-RBD P = 1.1 × 10–9) (Figure S9, SDC, https://links.lww.com/TXD/A470).

TABLE 3. - Characteristics of participant with Omicron-specific neutralizing response
NonresponderN = 24 ResponderN = 20 P
Male sex, N (%) 19 (79.2) 16 (80.0) 1
Age (y) at dose 3Median (IQR) 57.50 (51.75–65.25) 52.50 (41.25–58.25) 0.106
Transplant vintage at dose 3 (mo)Median (IQR) 58.62 (8.12–131.79) 24.73 (3.47–142.16) 0.316
eGFR (mL/min/1.73 m2)Median (IQR) 44.30 (36.05–63.27) 66.03 (47.92–79.18) 0.07
Donor type (%)
 Deceased donor 19 (79.2) 10 (50.0) 0.059
 Living donor 5 (20.8) 10 (50.0)
Immunosuppression at time of dose 3, N (%)
 Prednis1 23 (95.8) 18 (90.0) 0.583
 Antimetabolite (mycophenolate sodium/mofetil) 19 (79.2) 15 (75.0) 1
 CNI—tacrolimus 21 (87.5) 19 (95.0) 0.614
 CNI—cyclosporine 3 (12.5) 1 (5.0)
 On triple-agent immunosuppression 18 (75.0) 13 (65.0) 0.522
BMI (%)(median [IQR]) 23.91 (22.63–27.21) 27.89 (24.77–31.59) 0.392
Previous COVID-19 InfectionN (%) 2 (8.3) 0 (0.0) 0.493
3-dose vaccine series, N (%)
 Pfizer-BioNTech (BNT162b2) 21 (87.5) 14 (70.0) 0.208
 Moderna (mRNA-1273) 3 (12.5) 4 (20.0)
 Mixed 0 (0.0) 2 (10.0)
Dose and blood testing intervals (d)Median (IQR)
 Dose 1 to dose 2 21.00 (21.00–34.75) 21.00 (21.00–28.50) 0.593
 Dose 2 to dose 3 140.00 (122.50–173.75) 175.00 (130.25–192.75) 0.179
 Dose 3 to month 1 blood samples 25.00 (20.00–32.25) 28.00 (21.75–33.25) 0.322
BMI, body mass index; CNI, calcineurin inhibitor; estimated glomerular filtration rate; IQR, interquartile range.

TABLE 4. - Characteristics of participants with detectable neutralizing response to at least 1 variant
NonresponderN = 12 ResponderN = 32 P
Male sex, N (%) 10 (83.3) 25 (78.1) 1
Age (y) at dose 3Median (IQR) 58.00 (54.50–64.50) 53.00 (44.25–62.25) 0.158
Transplant vintage at dose 3 (mo)Median (IQR) 79.93 (23.99–131.79) 24.73 (3.47–142.16) 0.316
eGFR (mL/min/1.73 m2)Median (IQR) 44.30 (38.03–61.35) 55.91 (38.29–77.79) 0.171
Donor type (%)
 Deceased donor 9 (75.0) 20 (62.5) 0.5
 Living donor 3 (25.0) 12 (37.5)
Immunosuppression at time of dose 3, N (%)
 Prednis1 11 (91.7) 30 (93.8) 1
 Antimetabolite (mycophenolate sodium/mofetil) 11 (91.7) 23 (71.9) 0.241
 CNI—tacrolimus 11 (91.7) 29 (90.6) 1
 CNI—cyclosporine 1 (8.3) 3 (9.4)
 On triple-agent immunosuppression 10 (83.3) 21 (65.6) 0.459
BMI (%) (median [IQR]) 23.88 (23.36–26.66) 25.89 (24.19–29.86) 0.518
Previous COVID-19 infection, N (%) 1 (8.3) 1 (3.1) 0.476
3-dose vaccine series, N (%)
 Pfizer- BioNTech (BNT162b2) 11 (91.7) 24 (75.0) 0.677
 Moderna (mRNA-1273) 1 (8.3) 6 (18.7)
 Mixed 0 (0.0) 2 (6.3)
Third dose manufacturer, N (%)
 Pfizer-BioNTech (BNT162b2) 11 (91.7) 26 (81.2) 0.653
 Moderna (mRNA-1273) 1(8.3) 6 (18.8)
Dose and blood testing intervals (d)Median (IQR)
 Dose 1 to dose 2 21.00 (21.00–28.25) 21.00 (21.00–31.00) 0.896
 Dose 2 to dose 3 134.50 (120.75–151.25) 173.00 (133.25–196.50) 0.051
 Dose 3 to month 1 blood samples 23.50 (16.75–33.00) 26.50 (21.75–31.25) 0.413
BMI, body mass index; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate; IQR, interquartile range.

As anti-RBD and anti-spike IgG antibody levels at month 1 strongly correlated with neutralizing antibody levels (Log10ID50) at both months 1 and 3 (Figure S10, SDC, https://links.lww.com/TXD/A470), we next performed ROC analysis to identify the threshold values of anti-RBD and anti-spike antibodies at 1 mo following the third vaccine dose that were associated with the presence of neutralizing antibody against individual variants (Figure 5). For the WT, Beta, and Delta strains, the anti-RBD and anti-spike antibodies had comparable areas under the curve (AUCs), although the thresholds identified for anti-RBD were associated with zero false positives, in contrast to a minimal rate of false positives using the anti-spike threshold (Table S4, SDC, https://links.lww.com/TXD/A470). The AUCs were reduced in the case of Omicron (anti-RBD 0.91, anti-spike 0.925), with 3 false positives using the anti-RBD antibody threshold identified, and 4 false positives using the anti-spike antibody threshold identified.

F5
FIGURE 5.:
Threshold levels of binding antibody response associated with detectable neutralizing antibody. ROC analysis of anti-RBD and anti-spike antibody levels across (A) wild type, (B) Beta, (C) Delta, and (D) Omicron variants, for classification of the presence or absence of detectable neutralizing antibody (Log10ID50 >0). Areas under the curve (AUCs) for anti-spike (yellow) and anti-RBD (blue) are marked. Further details on the threshold values are found in Table S4 (SDC, https://links.lww.com/TXD/A470). RBD, receptor binding domain; ROC, receiver operating characteristic.

The optimal thresholds for anti-spike antibody levels that best identified neutralization capacities against all variants tested (WT: 1.319, Beta: 1.481, Delta: 1.391, and Omicron: 1.475) approximated the median convalescent antibody level seen in recovered healthy controls (1.38). In contrast, the anti-RBD antibody thresholds that best identified neutralization capacity against each variant (WT: 0.317, Delta: 0.627, and Beta: 0.932) were above the seropositivity threshold (0.186), but below the median convalescent response value of 1.25. Notably, in the case of Omicron, the optimal thresholds identified for both anti-spike (1.475) and anti-RBD (1.198) antibodies approximated or exceeded the median convalescent response, suggesting the requirement for a robust level of these antibodies as an identifier of Omicron neutralization capacity.

DISCUSSION

In this study, we comprehensively profiled the binding and neutralizing antibody responses up to 3 mo post-third mRNA SARS-CoV-2 vaccine dose in a KTR cohort. The majority of participants had detectable anti-spike and anti-RBD antibodies at 1 and 3 mo post-third vaccine dose. Our principal findings are (1) the proportion of patients whose antibody titers were consistent with a robust immune response rose significantly following a booster dose; (2) those who responded robustly at month 1 had a preserved humoral response at month 3; and (3) we define anti-RBD and anti-spike antibody levels that may aid in the identification of patients lacking neutralizing antibodies against Omicron, the current dominant variant worldwide.

Our observations regarding overall seropositivity following a third vaccine dose and seroconversion rates in previous nonresponders are largely in keeping with prior studies in SOTRs.12,14,15,17 Importantly, receipt of a third vaccine yielded a significantly increased proportion of patients with neutralizing capacity against WT, Beta, Delta‚ and Omicron variants after 1 mo; however, this response was inferior to that observed in healthy controls. Data on the development of an Omicron-specific neutralizing response among KTRs are limited, with rates of 12%39 and 43%40 reported at 1 mo. We detected Omicron-neutralizing antibodies in >45% of our cohort at 1 mo, with sustained response in 38.5% at 3 mo post-third mRNA-vaccine dose.

The optimum SARS-CoV-2 vaccination strategy in immunocompromised populations remains unclear. Our findings provide further evidence that the overall humoral response to a 3-dose vaccine regimen in KTRs remains inferior to the immunocompetent population, suggesting that alternative strategies, including further vaccine doses, immunosuppressive modulation, or use of complementary agents such as long-acting monoclonal antibodies or antivirals, may be necessary to induce a protective response against SARS-CoV-2.41–43 Concerningly, initial studies suggest that the “value-add” of a fourth vaccine dose may be limited in those with a poor response to a 3-dose vaccine series,43,44 with neutralization against Omicron largely unchanged.45 In a KTR cohort, only 10% of nonresponders after 3 doses achieved an adequate response following a fourth dose.46 These data suggest that further vaccine doses may prove insufficient to ensure protection from infection, and that an unidentified subset of vaccinated transplant recipients will remain at high risk.

There is a clear imperative to identify correlates of protection from which those with poor serological response could be readily identified. Studies conducted following 2-dose vaccination identified clinical factors including age, transplant duration‚ and use of mycophenolate or recent lymphocyte depletion therapies as associated with diminished binding antibody response.47–51 However, in both our study, and the recent study by Kumar et al31 in SOTRs, no demographic or transplant-related clinical characteristics emerged as significantly associated with an Omicron-specific neutralizing response at 1 mo post-third dose. In our cohort, those with detectable Omicron-neutralizing antibodies tended to have a higher eGFR and have received a living donor transplant (although these observations are likely linked). Improved vaccine response in proportion to eGFR is well-described amongst individuals with chronic kidney disease.52 Interestingly, patients with detectable neutralizing antibody against ≥1 variant tended to have a greater dosing interval between doses 2 and 3, although this did not reach statistical significance. A recent publication by Hall and Ferreira et al53 found that delayed-interval Pfizer-BioNTech (BNT162b2) mRNA SARS-CoV-2 vaccination was associated with enhanced humoral immune responses and robust T-cell responses in SOTRs.

Both humoral- and cell-mediated responses contribute to the development of protective immunity from SARS-CoV-2.54 The immune correlates of protection are not fully understood; however, binding and neutralizing antibody titers directly correlate with protection from SARS-CoV-2 infection in the general population.55–57 Functional T-cell responses are also used to infer the degree of immune response to SARS-CoV-2, yet difficulties with standardization and scalability of these assays have limited their widespread use beyond research settings.58 Although neutralizing antibody levels are highly predictive of the extent of immune protection from symptomatic SARS-CoV-2 infection,59 the assays required are typically cell-based, low-throughput, and resource intensive.60 In contrast, binding antibody assays are higher throughput and more readily scalable, and may thus represent the most accessible means of identifying the subset of patients who remain unprotected.61 The assay featured in our study uses publicly available reagents validated in 2 separate laboratories, in order to facilitate comparison of SARS-CoV-2 serology results between institutions.34 It is readily scalable, and has been used extensively in multiple patient populations.34,62,63 Our findings point to binding antibody thresholds, which may aid risk-stratification of patients, enabling prioritization of those requiring additional treatment strategies.

We acknowledge some limitations to this study. We could not evaluate the associations between neutralizing activity and clinical protection as there were no SARS-CoV-2 breakthrough infections in our population during the study period. Our relatively small cohort was male-predominant and may have been underpowered to detect clinical factors associated with an Omicron-specific neutralization response. Similarly, our study size precluded definitive assessment of the impact of mixed-vaccine regimens on the antibody response following 3-dose vaccination. However, our results, although underpowered, suggest that 3-dose mixed-vaccine or mRNA-1273-only regimens elicited stronger humoral responses, as has been reported amongst both hemodialysis patients and KTRs.35,64 Future, larger studies will be required to dissect this in greater detail. Although uncommon, some “false-positives” (suggesting the presence of neutralizing antibody) against Omicron BA.1 were present at the anti-RBD threshold identified in our analysis. Future studies are planned to further assess the discriminatory power of these binding antibody thresholds in a confirmatory cohort. Finally, the robustness of the protective immune response to SARS-CoV-2 is likely related to the combined activity of both humoral- and cell-mediated immunity, which was not investigated in this study.

In summary, ours is the first study to report on the durability of neutralizing antibodies to Omicron in a cohort of KTRs at 3 mo following the third vaccine dose. The majority of KTRs with anti-spike and anti-RBD antibody titers above median levels in convalescent serum had detectable neutralizing antibody. Although definitive immune correlates of protection are not fully elucidated, our work identifies binding antibody thresholds that may aid in the risk-stratification of patients. Importantly, quantitative binding antibody assays are relatively high throughput and scalable, making them a potentially attractive option worth further investigation.34

Although future studies will be required to explore the contribution of cell-mediated immunity, affinity maturation‚ and broadly neutralizing antibodies in this setting, as well as the impact of evolving Omicron subvariants, our data suggest that antibody levels may enable identification of vaccinated KTRs who remain highly susceptible to infection, and for whom additional therapies may be necessary.

ACKNOWLEDGMENTS

C.M.M. is supported by the Dr. Mary Papantony Nephrology research fund. K.T.A. is supported by a Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Award. A.C.G. is pillar lead for CoVaRR-Net and the Canada Research Chair (tier 1) in Functional Proteomics. This work was supported by funds from the COVID Immunity Task Force (CITF) (grant No. 2122-HQ-000071), which is funded by the Government of Canada, and the St. Michael’s Hospital Foundation. The robotics equipment used for the ELISA assays is housed in the Network Biology Collaborative Centre (NBCC) at the Lunenfeld-Tanenbaum Research Institute (ACG), a facility supported by Canada Foundation for Innovation funding, by the Ontario Government and by Genome Canada and Ontario Genomics (OGI-139). Sample intake and automated ELISA were performed by Geneviève Mailhot and Melanie Delgado Brand, and members of the NBCC, supervised by Karen Colwill, facilitated sample acquisition and data processing. We would like to thank the Nephrology Clinical Research team at St. Michael’s Hospital, Unity Health Toronto (Michelle Nash, Niki Dacouris, Lindita Dapi, Weiqiu Yuan, and Tiffany Thai) for collection and storage of study samples, and maintenance of the clinical research database. We would also like to thank the St. Michael’s Hospital Kidney Transplant Clinic staff for their support, and finally all of the kidney transplant recipients for their participation in this study. D.A.Y. was a CIHR New Investigator Award recipient and is the Canada Research Chair (Tier II) in Fibrotic Injury.

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