ANTI-A AND SARS-COV-2: AN INTRIGUING ASSOCIATION

Introduction: It is not clear which individual characteristics can determine susceptibility and intensity of symptoms, however, age, sex, ethnicity, hypertension and some haematological biomarkers, as D-dimer, thrombocytopenia and lymphopenia were associated with a worse outcome. Recently, it has been hypothesized that ABO blood groups can be related to susceptibility to the SARS-CoV-2 infection. Considering that the first studies reported A group as a risk factor and O group as a protection, some authors have been suggested that the anti-A antibodies, and not the blood group, could be responsible for the findings. Objectives: Based on these findings, this study analysed the association of SARS-CoV-2 infection with the presence (O and B blood groups type) or absence (A and AB blood groups type) of anti-A antibodies, considering the production of specific immunoglobulins (IgA, IgM and IgG) and neutralizing antibodies. Material and Methods: Retrospective study with 430 COVID-19 individuals (268 COVID-19 convalescent plasma donors-CCPD and 162 COVID-19 inpatients-CIP) from two Brazilian reference hospitals, confirmed by RT-PCR, and 2,212 healthy volunteer blood donors (VBD) as control group, that were evaluated and divided into two groups: with anti-A (O/B blood groups) and without anti-A group (A/AB blood groups). Immunoglobulins and neutralizing antibody titres were measured for CCPD and CID. Multivariate logistic regression and non-parametric tests were performed. Results: Although O blood group was the most frequent ABO group among VBD, A blood group was more frequent among COVID-19 individuals (CCPD 47.8%, CIP 43.2%, VBD 35.5%, p < 0.001). There was no statistical difference in blood groups distribution between CCPD and CIP (p=0.268). In our cohort, for each increased age year there was 6% more chance for COVID-19 (OR: 1.06;CI 95%: 1.05-1.06, p < 0.001), males showed 27% more chance for the disease (OR: 1.27;CI 95%: 1.02-1.59, p = 0.035) and O/B blood groups showed 38% less infection prevalence (OR: 0.62;CI 95%: 0.5-0.7, p < 0.001). Considering the fact that higher anti-A is usually described in O blood group, data from O versus B blood groups individuals were analysed and the former showed 34% less chance for COVID-19 (OR: 0.66;CI 95%: 0.46-0.95, p = 0.026). No difference regarding ABO group was found when COVID-19 inpatients of all blood types were analysed. Immunoglobulins A, M and G (IgA, IgM, and IgG) and neutralizing antibodies for SARS-CoV-2 were lower in COVID-19 individuals O/B blood groups (IgM p = 0.03, IgG p = 0.02, IgA p = 0.03). Discution: In our retrospective cohort, the COVID-19 individuals O/B blood groups (which produces anti-A) had 38% less chance to have a diagnosis of COVID-19 (p < 0.001) and the same groups showed lower titers of neutralizing antibodies, IgM, IgG and IgA. Groups O/B showed a protective factor against the SARS-CoV-2 infection, but it was not associated to COVID-19 inpatients (versus COVID-19 convalescent plasma donors) suggesting that blood type is not associated to SARS-CoV-2 infection severity. Conclusion: COVID-19 individuals from groups O/B showed lower titers of neutralizing antibodies, and IgM, IgG, and IgA lower levels.

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.

Anti-A and SARS-CoV-2: An intriguing association

Background/Case Studies: It is not clear which individual characteristics can determine susceptibility and intensity of symptoms, however, age, sex, ethnicity, hypertension and some haematological biomarkers, as Ddimer, thrombocytopenia and lymphopenia were associated with a worse outcome. Recently, it has been hypothesized that ABO blood groups can be related to susceptibility to the SARS-CoV-2 infection. Considering that the first studies reported A group as a risk factor and O group as a protection, some authors have been suggesting that the anti-A antibodies, and not the blood group, could be responsible for the findings. Study Design/Methods: A retrospective study with 430 COVID-19 individuals (268 COVID-19 convalescent plasma donors-CCPD and 162 COVID-19 inpatients-CIP) from two Brazilian reference hospitals, confirmed by RTPCR, and 2,212 healthy volunteer blood donors (VBD) as control group, that were evaluated and divided into two groups: one with anti-A (O/B blood groups) and one without anti-A group (A/AB blood groups). Immunoglobulins and neutralizing antibody titres were measured for CCPD and CIP. Multivariate logistic regression and nonparametric tests were performed. Results/Findings: Although O blood group was the most frequent ABO group among VBD, A blood group was more frequent among COVID-19 individuals (CCPD 47.8%, CIP 43.2%, VBD 35.5%, p<0.001). There was no statistical difference in blood groups distribution between CCPD and CIP (p=0.268). In our cohort, for each increased age year there was 6% more chance for COVID-19 (OR: 1.06;CI 95%: 1.05-1.06, p<0.001), males showed 27% more chance for the disease (OR: 1.27;CI 95%:1.02-1.59, p=0.035) and O/B blood groups showed 38% less infection prevalence (OR: 0.62;CI 95%: 0.5-0.7, p<0.001). Considering the fact that higher anti-A is usually described in the O blood group, data from O versus B blood groups individuals were analysed and the former showed 34% less chance for COVID-19 (OR: 0.66;CI 95%:0.46-0.95, p=0.026). There was no difference regarding ABO group found when COVID-19 inpatients of all blood types were analysed. Immunoglobulins A, M and G (IgA, IgM and IgG) and neutralizing antibodies (NAbs) for SARS-CoV-2 were lower in COVID-19 individuals O/B blood groups (NAbs p=0.008, IgM p=0.03, IgG p=0.02, IgA p=0.03). Conclusions: In our retrospective cohort, the COVID-19 individuals O/B blood groups (which produces anti-A) had 38% less chance to have a diagnosis of COVID-19 (p<0.001) and the same groups showed lower titers of neutralizing antibodies, IgM, IgG and IgA. Groups O/B showed a protective factor against the SARS-CoV-2 infection, but it was not associated to COVID-19 inpatients (versus COVID-19 convalescent plasma donors) suggesting that blood type is not associated to SARSCoV- 2 infection severity.

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.