Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants

Abstract

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population.

Keywords: COVID19; SARS‐CoV‐2; cellulose pulldown assay; humoral response against COVID19 variants; neutralizing antibody; point‐of‐care test; serology test.

FIGURE 1

Schematic of cpVNT workflow and results obtained with contrived whole‐blood sample. (a) Graphical representation of the modified cpVNT workflow before detection in fluorescent reader. (b) Cartoon depicting of possible molecular events occur in samples with and without nAb on the test spot and reagent control spot. (c) Measurement of fluorescence intensity from prevaccination whole blood samples titrated with 1, 5, 10, 25, 50, 100 nM SARS‐CoV‐2 monoclonal neutralizing antibody (nAb). (d) The percent blocking calculated from Equation (1) with nAb concentration presented in log scale. cpVNT, cellulose pulldown virus neutralization test; nAb, neutralizing antibody

FIGURE 2

Evaluation of nAb response among prevaccination and postvaccinated individuals using modified cpVNT. (a) The percent blocking measured from 170 whole blood samples of prevaccination, Pre‐Vac (n = 36), post first dose (1–2W n = 10, 3–6W n = 50) and post second dose (n = 74). The gray line is the median from each group while each dot represents the mean from three independent experiments. (b) The nAb percent blocking measured in individuals opt for BNT162b2 (n = 4 from P1 1–2W, n = 30 from P1 3–6W, n = 40 from P2) or (c) mRNA‐1273 (P1 1–2W n = 6; P1 3–6W n = 20; P2 n = 34). Kruskal–Wallis test with Dunn's multiple comparison was performed between each vaccination status. (d) Comparison of nAb percent blocking at P1 3–6W BNT162b2 and P1 3–6W mRNA‐1273. Two‐tailed Mann–Whitney test was performed between the two vaccine brands. The significance values *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Total samples: Pre‐Vac versus P1‐3–6W, p < 0.001; Pre‐Vac versus P2, p < 0.0001. BNT162b2: Pre‐Vac versus P1‐3–6W, p < 0.05; Pre‐Vac versus P2, p < 0.0001. mRNA‐1273: Pre‐Vac versus P1‐3–6W, p < 0.001; Pre‐Vac versus P2, p < 0.0001. (e) Twenty‐two individual samples percent blocking mapped over pre and post vaccination period comparing between two types of vaccines, BNT162b2 n = 12, mRNA 1273 n = 10. The window period between first and second dose of vaccination ranged from 4 to 6 weeks depending on individual's choice. cpVNT, cellulose pulldown virus neutralization test; nAb, neutralizing antibody

FIGURE 3

Comparison of modified cpVNT with international standards and established serology tests. (a) The performance of the First WHO International Standard Anti‐SARS‐CoV‐2 immunoglobulin (20/136), Reference Panel for anti‐SARS‐CoV‐2 Mid‐tire and Low‐titre plasma using modified cpVNT. (b) The correlation of percent blocking measured from 30 matching plasma and blood samples at prevaccination (Pre‐Vac), post first dose (P1) and post second dose (P2) phase using modified cpVNT gave Pearson r, 0.908. The percent blocking for cpVNT with blood samples that correspond to 1000, 220, and 44 IU/ml are determined by assuming a linear correlation between the two sample types (see accompanying table). (c) Comparison of percent blocking measured in the modified cpVNT with percent inhibition of sVNT (cPass) in 44 matching Pre‐Vac, P1, and P2 venous blood and plasma samples. The sensitivity was calculated as 81.5% (CI: 61.9%–93.7%), and specificity is 100% (CI: 81.5%–100%) when both cpVNT and sVNT's (cPass) thresholds were set at 30% blocking. (d) Comparison between cpVNT and pseudovirus neutralization test (pVNT) with 20 individuals' sample. The pVNT was performed with plasma in 1:80 dilution. The sensitivity is 100% (CI: 47.8%–99.9%) and specificity is 66.7% (CI: 38.4%–88.2%) with 30% blocking as the threshold for cpVNT and 50% neutralization for pVNT. All experiments were performed in triplicates. CI, confidence interval; cpVNT, cellulose pulldown virus neutralization test; nAb, neutralizing antibody; pVNT, pseudovirus neutralization test

FIGURE 4

Assessment of nAb response to variants of concern depending on vaccine brand or time postvaccination with our modified cpVNT. (a) Whole blood samples (n = 39) from participants who completed vaccination within 3 months were tested with wild‐type (WT) RBD‐CBD and variant RBD‐CBD. Friedman test with multiple comparison was performed comparing the variants against WT, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. WT versus alpha, p < 0.01; WT versus beta, p < 0.0001; WT versus gamma, p < 0.0001; WT versus delta, n.s. (b). The percent blocking of nAb measured from 20 P2 samples of BNT162b2 recipients (≤3 months) when tested with RBD‐CBD variants. (c) Similarly, percent nAb blocking of 19 P2 samples (≤3 months) from mRNA‐1273 recipients tested with the RBD‐CBD variants. (d) The nAb response in BNT16b2 recipients against WT and RBD‐CBD variants when comparing two groups: within 3 months (n = 20) and more than 3 months (n = 37) after complete vaccination. Two‐sided Mann–Whitney test was performed between the two groups for each variant, WT p < 0.0001, alpha p < 0.001, beta p < 0.0001, gamma p < 0.001, delta p < 0.0001. CBD, cellulose binding domain; cpVNT, cellulose pulldown virus neutralization test; nAb, neutralizing antibody; RBD, receptor binding domain