Screening for SARS-CoV-2 antibodies in convalescent plasma in Brazil: Preliminary lessons from a voluntary convalescent donor program

Abstract

Background: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) collection began in two Brazilian hospitals for treatment of severe/critical patients.

Methods and materials: Mild/moderate COVID-19 convalescents were selected as CCP donors after reverse transcription polymerase chain reaction (RT-PCR) confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and absence of symptoms for ≥14 days plus (a) age (18-60 years), body weight greater than 55 kg; (b) immunohematological studies; (c) no infectious markers of hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T-lymphotropic virus-1/2, Chagas and syphilis infection; (d) no HLA antibodies (multiparous); (e) second RT-PCR (nasopharyngeal swab and/or blood) negativity; (f) virus neutralization test (cytopathic effect-based virus neutralization test neutralizing antibody) and anti-nucleocapsid protein SARS-CoV-2 IgM, IgG, and IgA enzyme-linked immunosorbent assays.

Results: Among 271 donors (41 females, 230 males), 250 presented with neutralizing antibodies. Final RT-PCR was negative on swab (77.0%) or blood (88.4%; P = .46). Final definition of RT-PCR was only defined at more than 28 days after full recovery in 59 of 174 (33.9%) RT-PCR -ve, and 25/69 RT-PCR +ve (36.2%; 13 between 35 and 48 days). Neutralizing antibody titers of 160 or greater were found in 63.6%. Correlation between IgG signal/cutoff of 5.0 or greater and neutralizing antibody of 160 or greater was 82.4%. Combination of final RT-PCR -ve with neutralizing antibody ≥160 was 41.3% (112/271). Serial plasma collection showed decline in neutralizing antibody titers and IgA levels (P < .05), probably denoting a "golden period" for CCP collection (≤28 days after joining the program); IgA might have an important role as neutralizing antibody. Donor's weight, days between disease onset and serial plasma collection, and IgG and IgM levels are important predictors for neutralizing antibody titer.

Conclusions: RT-PCR +ve cases are still detected in 36.2% within 28 to 48 days after recovery. High anti-nucleocapsid protein IgG levels may be used as a surrogate marker to neutralizing antibody.

Keywords: COVID-19; SARS-COV-2; coronavirus; convalescent plasma therapy; passive immune therapy.

FIGURE 1

CCP program stages. All donors with a previous RT‐PCR1 positive had their first medical interview on the average 33.4 ± 7.9 (range, 14‐65) days after onset of symptoms, which lasted 10.8 ± 5.2 (range, 0‐33) days. Main symptoms are shown in upper left square.). Only one donor was asymptomatic (RT‐PCR1 +ve), due to close contact with a confirmed patient. If RT‐PCR2 was positive, donors were asked to collect another RT‐PCR within 14 days. If RT‐PCR3 negative, they were accepted in the program; otherwise, they were discarded. Accepted donors with multiple collection (apheresis) are displayed, based on the mean ± SD (range) of days after full recovery

FIGURE 2

Distribution of 271 convalescent donors, based on nasopharyngeal swab (NF swab, n = 139) or peripheral blood RT‐PCR (PB, n = 121), and neutralizing antibody tests (n = 250). Eleven (11) donors did not collect RT‐PCR2. There were 107 donors who were tested by both PB and swab RT‐PCR2, whose cycle threshold (mean ± SD) was, respectively, 37 ± 1.3 × 36 ± 1.8 (P = .34, Wilcoxon test). The final percentage of accepted donors according to the combination of RT‐PCR2 or 3 and neutralizing antibody titer of 80 or higher or 160 or higher is 49.1% (95% CI, 43.0%‐55.2%) or 41.3% (95% CI, 35.4%‐47.4%), respectively (middle box)

FIGURE 3

Length of days after full recovery (absence of any symptoms referred by the donors) from 243 CCP donors who had a full definition of final RT‐PCR status (RT‐PCR2 or 3), given as mean (±SD; range). Long dashed vertical line marks the 28‐day period after full recovery, where several regulatory agencies consider safe to donate blood once COVID‐19 symptoms have vanished. ³⁵ , ⁴⁷ , ⁴⁸ Upper left and right figures show donors tested only by nasopharyngeal swab (n = 136) or peripheral blood (n = 107) RT‐PCR, respectively. The combination of both methods is shown in the lower figure, with a total of 33.9% (n = 59/174) and 36.2% (25/69) donors, respectively, for RT‐PCR –ve and RT‐PCR +ve, who had their final PCR status defined after the 28‐day period after full recovery

FIGURE 4

Neutralizing antibody titers from 250 donors (male = 218; female = 32). Titers <20 denote a negative reaction (absence of nAb). Lower and upper arrow indicates number (percentage) of donors with titers <80 and <160, respectively, which were not accepted as CCP donors, based on the chosen cutoff

FIGURE 5

Immunoglobulin anti‐NP (IgM, and IgA, left; IgG right) S/CO according to ln nAb titers from 226 convalescent plasma with final RT‐PCR status. The horizontal and vertical long‐dashed lines on the right upper quadrant (IgG) denotes a proposed limit for both high IgG S/CO level (≥5.0; above the horizontal dashed line) and neutralizing antibody titer ≥160 (right of vertical dash line). Agreement between IgG S/CO ≥5.0 and neutralizing antibody ≥160 was 82.4%. There is a strong correlation between immunoglobulin levels (S/CO) and neutralizing antibody titers (ln), as measured by the Spearman's rho correlation coefficient (0.49, 0.66, and 0.54 for IgM, IgG, and IgA, respectively; all P < .01)

FIGURE 6

Decline in neutralizing antibody titers (ln) and IgA (S/CO) from 52 CCP donors who donated at least twice by plasmapheresis. Horizontal line shows the mean for each marker. There is a median interval of 14 days between collection of screen sample and Collection 1, and additional 8 days (median) between Collections 1 and 2. Left: neutralizing antibody titers (ln); right: IgA (S/CO). There is a statistically significant decline for nAb for all periods (screen, Collections 1 and 2; all P < .05). For IgA, statistical difference was seen for screen sample × Collection 1 (P = .01) and screen sample × Collection 2 (P = .05). There was no statistically significant decline for IgM and IgG. All results obtained by Wilcoxon test

FIGURE 7

Added variable plot derived from a stepwise multiple regression between neutralizing antibody titers (ln) and the key variables found to exert main dependence on neutralizing antibody: Weight (kg); time since onset of illness (days); IgG and IgM (S/CO). The y‐axis ‐ e (lnacneutral | X) ‐ shows the relationship between neutralizing antibody and each independent variable (x‐axis), adjusting for the effects of other remaining independent variables. ⁴⁷ There is a mild, negative effect just for the onset of illness in days (P = .09). All the remaining independent variables are statistically significant. The model follows the equation: Y = 2.585 + 0.017b ‐ 0.017c + 0.185d + 0.262e, where Y = neutralizing antibody titer (ln); b = weight (kg); c = onset of illness (days); d = IgG S/CO; e = IgM S/CO