Convalescent plasma transfusion for COVID-19: Impact of neutralizing antibodies on respiratory outcomes

Background/Case Studies: In the past months, convalescent plasma (CP) has been used as an alternative therapy to treat COVID-19 patients. Previous studies highlighted the role of neutralizing antibody titers (NAbs) on clinical improvements. We analysed a series of cases of COVID patients treated with CP transfusion and associations between transfused NAbs and patient NAbs on respiratory outcomes. Study Design/Methods: Thirty patients diagnosed with SARS-CoV-2 by RT PCR and severe pneumonia were prospectively analysed at a single center in Brazil. Doses of 300-600ml of CP were transfused. To assess respiratory outcomes, PaO2/FiO2 ratios were determined at Day 0 (day of CP transfusion), Day 5 and by discharge and duration of ventilation support were analysed. Improvements were determined by variations of PaO2/ FiO2 ratios from Day 0 through day 5 (V0-5) and from Day 0 through discharge (V0-D). Neutralizing antibodies from patients prior to transfusion (NAbsP) and neutralizing antibodies of CP units transfused (NAbsCP) were analysed. NAbsCP considers the total amount of antibodies transfused to account for volume differences. We performed a generalized estimating equations (GEE) approach with logit link for binary data to model the effect of Nabs CP and variations on PaO2/FiO2 ratios between Day 0-Day 5 and Day 0-discharge. Regression models were performed to determine the predictive variables associated to improvements on PaO2/FiO2 ratios and duration of ventilation support. Results/Findings: Variations of PaO2/FiO2 ratios from day 0 through Day 5 and day 0 through discharge are displayed on Table 1. Significant improvements on PaO2/ FiO2 ratios were observed from Day 0 through discharge (p<0.001). NAbsP were associated to higher improvements on PaO2/FiO2 on V0-D (mean difference (MD)): 2.8 CI 95% -1.0-4.6 p 0.003), NAbsCP, however, were associated to minor variations in PaO2/FiO2 (MD -4.8 CI95% -7.9;-1.7 p 0.002) on the same interval. At each 100 unit increase in NAbs CP, variations on PaO2/FiO2 on V0-D were expected to be 4.8 units lower. When analysing V0-5, NAbsCP were again associated to minor improvements of PaO2/FiO2 ratios from D0 through D5 (MD -3.6 95%CI -7.2;-0.003 p 0.05). Other variables did not show statistical significance. When considering duration of ventilation support neither NabsP (Mean Ratio MR 0.985 CI 95% 0.962-1.007 p 0.185) nor NAbsCP (MR 0.967 CI95% 0.918 1.018 p 0.198) showed significant statistical differences. Conclusions: In our analysis, NAbs produced from the patient prior to CP transfusion are associated to better improvements on respiratory outcomes when compared to NAbs from transfused units. Regarding duration of mechanical ventilation, neither NAbsP nor Nabs CP had impact on outcomes.

Predictive factors for intensive care unit length of stay in COVID-19 patients treated with convalescent plasma

Background/Case Studies: Convalescent plasma (CP) has emerged in the past months as an alternative treatment for COVID-19. We prospectively analysed the outcomes of critically ill COVID-19 patients treated with CP in order to investigate predictive factors of intensive care unit (ICU) length of stay at a single center in Brazil. Study Design/Methods: Thirty COVID-19 patients laboratory-confirmed by RTPCR with severe pneumonia were recruited at a single center in Brazil. Doses of 300-600ml of CP were administered. Primary outcomes were ICU length of stay and duration of mechanical ventilation support.The following variables were analysed: Severity organ failure assessment score(SOFA) at day 0 (day of CP transfusion), patient ABO blood group, concomitant use of any other therapies (hydroxychloroquine, azythromicin, tocilizumab, immunoglobulin), neutralizing antibody titers of the patients prior to transfusion (NAbsP) and total titers of neutralizing antibody from CP units transfused (NAbsCP). NAbsCP considers the total amount of antibodies transfused to account for volume differences. Multivariate logistic regression was performed. Results/Findings: In our case series, SOFA at day 0, ABO group, and both NAbs P and NAbsCP were predictive factors of ICU length of stay. At each point increased in SOFA, ICU length of stay was 38.7% longer (MR: 1.387 CI 95% 1.254-1.534, p<0.001). Group A had36.1% longer ICU length of stay (MR: 0.639 CI 95% 0.417-0.980, p 0.04). Increases of 100 units in NAbsP resulted in 1% reduction of ICU lenght of stay (MR 0.990 CI 95% 0.982-0.998, p 0.017). Similar results were obtained with NAbs-CP, with 2% reduction in ICU length of stay (MR 0.980 CI95% 0.968-0.993, p 0.002) at each 100 unit increase. Use of other therapies did not influence ICU lenght of stay (p 0.373). We perform the same analysis for duration of mechanical ventilation support, and all the variables failed to demonstrate any association Conclusions: Our findings suggest that severity of disease prior to transfusion, ABO group, patient capacity to produce neutralizing antibodies and transfusion of CP with high titer NAbs are significant predictive factors for ICU length of stay. High titer NAbs CP may add benefit to these patients. No association was found between these same variables and duration of mechanical ventilation.

Screening for SARS-CoV-2 antibodies in convalescent plasma (CCP) in Brazil: Results from a voluntary convalescent donor program

Background/Case Studies: We describe the COVID- 19 Convalescent Plasma (CCP) program from two Brazilian hospitals for treatment of severe/critical patients Study Design/Methods: Mild/moderate COVID-19 convalescents were selected as CCP donors after RT-PCR confirmed SARS-CoV-2 infection and absence of symptoms for ≥14 days plus: 1) age (18-60yrs), body weight >55kg;2) immunohematological studies;3) no infectious markers of HBV, HCV, HIV, HTLV1-2, Chagas and syphilis infection;4) no HLA antibodies (multiparous);5) Second RT-PCR (nasopharyngeal swab and/or blood) negativity;6) virus neutralization test (CPE-based VNT nAb) and anti-nucleocapsid (NP) SARS-CoV-2 IgM, IgG and IgA ELISAs. Results/Findings: Among 271 donors (41 females, 230 males), 250 presented with nAb. Final RT-PCR was negative on swab (77.0%) or blood (88.4%;p= 0.46). Final definition of RT-PCR was only defined at>28days after full recovery in 59/174 (33.9%) RT-PCR-ve, and 25/69 RT-PCR+ve (36.2%;13 between 35-48 days). NAb titers ≥160 was found in 63.6%. Correlation between IgG signal/ cut-off ≥5.0 and nAb ≥160 was 82.4%. Combination of final RT-PCR-ve with nAb ≥160 was 41.3% (112/271). Serial plasma collection showed decline in nAb titers and IgA levels (p<0.05), probably denoting a “golden period” for CCP collection (≤ 28 days after joining the program);IgA might have an important role as nAb. Donor's weight, days between disease onset and serial plasma collection, IgG and IgM levels are important predictors for nAb titer. Conclusions: RT-PCR+ve cases are still detected in 36.2% within 28-48 days after recovery. High anti-NP IgG levels may be used as surrogate marker to nAb.

Kinetics of neutralizing antibodies (nAb) SARS-CoV-2 and anti-nucleocapsid (NP) antibodies in convalescent plasma donors

Background/Case Studies: COVID-19 convalescent plasma (CCP) has been used for therapy in severely affected COVID-19 patients. The rational relies on the presence of nAb in convalescent's bloodstream, which might suppress patient's viremia. Little is known about the nAb kinetics in CCP donors. Study Design/Methods: A cohort of previously RTPCR+ ve, male, volunteer, non-remunerated, mild convalescent donors has been studied, based on serial virus neutralization tests (CPE-based VNT, GenBank: MT MT350282, transformed in natural log) and specific IgM, IgG and IgA anti-nucleocapsid protein (NP) SARS-CoV-2 ELISA, depicted as signal/cutoff (S/CO). Results/Findings: A total of 63 donors were evaluated within a period ranging from 14-97 days after full recovery of symptoms (DARS). There was initially a decline in nAb and IgA anti-NP from the first to third collection (median = 45days), followed by an unexpected rise in two additional collections. No differences were seen for IgM and IgG anti-NP. Data are shown below, with statistical values between subsequent samples. Conclusions: There is a great variability in nAb titers, with a declining trend over time. Although this was clear during the first three collections, the sudden rise could be explained by biological nAb fluctuation or by viral re-exposure after recovery, due to contact with infected people (pandemic still active in our region). Although IgA anti-NP shows a wide range, its declining trend could be signaling a possible role of IgA as an important component of nAb. Further studies are required to better understand the kinetic behavior of these antibodies.

Preservation of SARS-CoV-2 neutralizing antibodies (nAb) or anti-nucleocapsid proteins (NP) in convalescent donor plasma (CCP) Treated with Amotosalen/UVA Illumination (A/UVA)

Background/Case Studies: COVID-19 Convalescent plasma (CCP) has been used for therapy in severely symptomatic COVID-19 patients. Pathogen reduction (PR) has been proposed to mitigate the risk of transfusion-transmitted infectious agents. We investigate the impact of A/UVA on nAbs and anti-NP (IgM, IgG and IgA) PR treatment of CCP units. Study Design/Methods: Plasmapheresis CCP units (600 mL) were collected from a cohort of previously confirmed male RT-PCR positive [+ve] COVID-19 mild/ moderate convalescent patients, all first-time and nonremunerated volunteers, with >14 days after full recovery of symptoms. CCP units were treated with INTERCEPT Blood System (Cerus Corporation, Concord, USA) according to manufacturer's instructions, either individually or pooled two by two. After treatment, units were separated into 200 mL doses. Pre- and post-PR treatment samples were harvested and kept at 4oC for 3-5 days prior to testing for nAb titers using a CPE-based virus neutralization assay (GenBank: MT MT350282), and specific IgM, IgG and IgA anti-NP antibodies by ELISA. Results/Findings: A total of 16 individual and 94 pooled units were treated (n =110 CCP donations), rendering 330 x 200 mL treated CCP therapeutic doses. There were no statistical differences in samples harvested before versus after A/UVA treatment (all p>0.05, Wilcoxon test) for nAb titers or IgM, IgG and IgA anti-NP absorbance levels, as shown in the table. Conclusions: Anti-NP IgM, IgG, IgA, and nAbs are not adversely impacted by A/UVA treatment, suggesting this PR technology can be employed to mitigate the risk of transfusion-transmitted infections after collection of CCP donors, who are often first time blood donors. With most CCP units destined to treat older, immunosuppressed patients with several comorbidities, the use of A/UVA PR treatment is not only safe and recommended, while preserving anti-SARSCoV- 2 antibodies in CCP units.

CINÉTICA DE ANTICORPOS NEUTRALIZANTES (NAB) E DE LIGAÇÃO CONTRA ANTÍGENOS NUCLEOCAPSÍDEOS (NP) DE SARS-CoV-2 EM DOADORES DE PLASMA CONVALESCENTE (CCP)

Objetivos: O plasma convalescente COVID-19 (CCP) tem sido usado como terapia em pacientes graves desta infecção. O príncipio racional baseia-se na presença de anticorpos neutralizantes (nAb) na circulação de pacientes convalescentes, podendo assim, suprimir a viremia em receptores. A cinética destes nAb ainda é pouco conhecida até o momento. Materiais e métodos: Uma coorte de doadores convalescentes, todos RT-PCR+vo, masculinos e voluntários tem sido acompanhada por meio de coletas seriadas para testes de nAb (teste de neutralização viral – CPE-based VNT, GenBank: MT MT350282, cujos títulos foram transformados em logaritmo natural) e por anticorpos de ligação IgM, IgG e IgA específicos contra proteínas do nucleocapsídeo (NP) – SARS-CoV-2 ELISA – apresentados sob a forma de relação absorbância/cut-off, (S/CO). Resultados: Até o momento, 78 indivíduos foram avaliados, dentro de um período entre 14–97 dias após o término completo de sintomas (TCS). Observou-se inicialmente um declínio nos títulos de nAb (403±3 × 221±3, p=0.004) e IgA anti-NP (S/CO = 2.9±4.2 × 2.4 ±3.5, p=0.04) da primeira à terceira coleta sérica (mediana = 45 dias), seguida por uma súbita e inesperada elevação após duas coletas adicionais. Não foram observadas alterações estatisticamente significativas para os níveis de IgM e IgG anti-NP. Conclusão: Existe uma grande variabilidade nos títulos de nAb, com uma tendência de declínio ao longo do tempo. Embora este fenômeno seja evidente durante as três primeiras coletas, a súbita elevação pode ser talvez explicada por flutuações biológicas dos títulos, ou por reexposição viral após a recuperação destes indivíduos, devido ao contato ativo com outras pessoas infectadas, posto que ainda estão vivendo em ambientes acometidos pela pandemia atual. Embora a IgA anti-NP demonstre uma ampla variação, sua tendência ao declínio pode sinalizar um possível papel da IgA como importante componente de nAb. Mais estudos são necessários para o entendimento do comportamento cinético destes anticorpos.