Anti–SARS-CoV-2 Antibody Responses in Convalescent Plasma Donors Are Increased in Hospitalized Patients; Subanalyses of a Phase 2 Clinical Study

We evaluated the antibody responses in 259 potential convalescent plasma donors for Covid-19 patients. Different assays were used: a commercial ELISA detecting antibodies against the recombinant spike protein (S1); a multiplex assay detecting total and specific antibody isotypes against three SARS-CoV-2 antigens (S1, basic nucleocapsid (N) protein and receptor-binding domain (RBD)); and an in-house ELISA detecting antibodies to complete spike, RBD and N in 60 of these donors. Neutralizing antibodies (NAb) were also evaluated in these 60 donors. Analyzed samples were collected at a median time of 62 (14–104) days from the day of first symptoms or positive PCR (for asymptomatic patients). Anti-SARS-CoV-2 antibodies were detected in 88% and 87.8% of donors using the ELISA and the multiplex assay, respectively. The multivariate analysis showed that age ≥50 years (p < 0.001) and need for hospitalization (p < 0.001) correlated with higher antibody titers, while asymptomatic status (p < 0.001) and testing >60 days after symptom onset (p = 0.001) correlated with lower titers. Interestingly, pseudotype virus-neutralizing antibodies (PsNAbs) significantly correlated with spike and with RBD antibodies by ELISA. Sera with high PsNAb also showed a strong ability to neutralize active SARS-CoV-2 virus, with hospitalized patients showing higher titers. Therefore, convalescent plasma donors can be selected based on the presence of high RBD antibody titers.

(v) two negative SARS-CoV-2 PCR results (nasal and/or pharyngeal swap); the time interval between the two negative tests should be at least 7 days; (vi) the second negative test should be at least 7 days prior to the plasmapheresis; (vii) male donors without transfusion history and females without history of transfusion or pregnancy; (viii) normal full blood count; (ix) negative serological tests for HBV, HCV, HIV, VDRL, HTLV-1 and negative for HIV, HBV, HCV (with NAT); (x) donors should fulfill the general criteria for blood donation: age 18-60 for first time donors, age up to 65 for multiple times donors, body weight >=50 kg, good general condition, Hb >12.5 g/dL for female, Hb >13.5 g/dL for male donors; (xi) Total serum protein ≥60 g/l; (xii) normal IgG levels; (xiii) arterial blood pressure 100-180 mmHg, diastolic<100 mmHg; (xiv) heart rate 50-110/min (for athletes a heart rate <50/min was accepted); (xv) normal body temperature.
Data collection for plasma donors: In order to correlate the level of anti-SARS-CoV-2 antibodies to the severity of the disease the following donor information was collected: (i) time of initial symptoms of COVID-19 infection; (ii) time of positive PCR result of nasal/pharyngeal swab for SARS-CoV-2; (iii) disease symptoms (fever, fatigue, diarrhea, dyspnea, loss of smell, loss of taste, etc); (iv) organs involved in the infection as determined by clinical examination, laboratory or imaging tests (i.e. lung CT scan); (v) time from diagnosis to full recovery of symptoms of the disease; (vi) time from diagnosis to the first and second negative PCR result of nasal/pharyngeal swab for SARS-CoV-2; (vii) need for hospitalization during COVID-19 infection; (viii) need for oxygen supply during the infection; (ix) need for ICU admission or for mechanical ventilation.

RNA Extraction and Real Time RT-PCR for SARS-CoV-2: Please see
supplementary file Nasopharyngeal and/or oropharyngeal swabs were collected and transferred to the Microbiology laboratory, immersed in an appropriate virus transport medium (e.g. UTM Viral Transport, Copan Diagnostics Inc., Brescia, Italy).
Automated purification of viral RNA from viral transport medium was performed using the QIAsymphony DSP virus/pathogen mini kit on the QIAsymphony SP platform (QIAGEN, Hilden, Germany). A Real Time, one -step Reverse Transcription -PCR, specific for ORF1ab gene of SARS-CoV-2 is finally performed using the VIASURE SARS-CoV-2 Real Time PCR Detection Kit (CerTestBiotec SL, Zaragoza, Spain).

Detection of anti-SARS-CoV2 antibodies using Euroimmun Elisa: Both assays for
IgG and IgA antibodies against the S1 domain of the virus were performed on the automated EUROIMMUN Analyzer I (Euroimmun Medizinischem Labordiagnostika AG, Lubeck, Germany), according to the manufacturer's protocol. The anti-SARS-CoV-2 IgG ELISA has 90% sensitivity (95% CI: 74.4-96.5) and 100% specificity (95% CI: 95.4-100), according to manufacturer. The perfromance of the Euroimmun assays for the determination of IgG and IgA antibodies against the SARS-CoV-2 virus was evaluated with sera from patients with COVID-19, hospitalized in Greek hospitals. The infection was confirmed with real time PCR. Only sera collected after the 10 th day since the appearance of the symptoms were included. The clinical sensitivity and the 95% CI was 91% (83-97) for the IgG and 97.5% (93-99) for the IgA assays, respectively. The specificity evaluated with sera collected before 2020 was 97.9% (83-100) for the IgG and 90% (68-98) for the IgA assays, respectively.

Detection of anti-SARS-CoV2 antibodies using ProtATonce Multiplex Assay:
Details of the assay development and testing can be found at https://www.medrxiv.org/content/10.1101/2020.09.09.20191122v2. In summary, antigens were covalently coupled to color-coded magnetic Luminex microspheres with distinct fluorescent signatures (bead regions) allowing the simultaneous detection of antibody responses to the different antigens from the same serum sample.
The SARS-CoV-2 N and S1 antigens were purchased from the Native Antigen criterion for antibody presence from N, RBD, and S1 results is based on the optimal logical rule of antigens [RBD OR (S1 AND N)] that is also depicted as a truth