PMC8278474_01
Patient outcome from symptoms onset to hospital discharge.
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Care Unit of the same hospital because of severe respiratory distress secondary to COVID-19 that, after a short unsuccessful trial of Continuous Positive Airways Pressure (CPAP), required intubation for invasive mechanical ventilator support ( Figure 1 ). Clinical course was complicated by a urinary tract infection and an Aspergillus super-infection of the lower respiratory tract that fully recovered with specific antimicrobial therapy. Quantitative real time polymerase chain reaction (qPCR) failed to detect SARS-CoV-2 nucleic acid in two nasopharyngeal swabs, but qPCR detection of SARS-CoV-2 RNA in a bronco-alveolar lavage (BAL) performed on June 14, after transfer to a sub-intensive care unit, confirmed viral invasion of lower respiratory tract ( Figure 1 ).
Levels of anti-SARS-CoV-2 N IgG antibodies in the concentrate antibody solution infused to the patient, in antibody donor serum and in recipient sera collected before antibody infusion (day 0) and 7, 14, and 22 days thereafter. The dashed line indicates the threshold for antibody positivity.
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Seven hundred milliliter of a saline solution of anti-SARS-CoV-2 N IgG (Index 8.8,  Figure 2 ) were obtained (after filter washing at the end of the DFPP session) from a 61-year-old male ABO competent adult donor who, according to the clinical and laboratory criteria issued by Consiglio Superiore di Sanita on February 28, 2020, had definitely recovered from COVID-19. The donor was found to have an adequate titer of anti-SARS-CoV-2 S1/S2 (84 UA/ml) and anti-N IgG (7.74 index,  Figure 2 ). SARS-CoV-2 S1/S2 IgG test (DiaSorin) and subsequently confirmed by chemiluminescence microparticle immunoassay (CMIA) Alinity SARS-CoV-2 IgG (Abbott) detection of anti-nucleocapsid antigen (N) circulating IgG. Serum levels of anti-SARS-CoV-2 N IgG antibodies, which were almost undetectable before antibody infusion, sharply increased at post-infusion day 1 and then slowly and progressively declined at post-infusion days 7, 14, and 21 ( Figure 2 ). However, following the infusion antibody titer largely exceeded the upper limit for test positivity (index 1.2) up to day 21 ( Figure 2 ).
Patient serum-induced ex vivo C5b-9 deposition (A) and thrombi formation (B) on cultured Human Microvascular Endothelial Cells (HMEC-1) line immediately before antibody infusion (Day 0), and 1, 7, 14, and 21 days thereafter. (A) HMEC-1 were incubated for 2 h with serum (diluted 1:2 with test medium, HBSS with 0.5% BSA) from the patient or with a control serum pool. At the end of incubation, cells were washed, fixed, and stained with rabbit anti-human complement C5b-9 complex antibody followed by FITC-conjugated secondary antibody. Fluorescence microscopy was used to view the fluorescent staining on endothelial cell surface, and the HMEC-1 area covered by C5b-9 staining was calculated by automatic edge detection (Image J software) in 15 high power fields. For each sample, the highest and the lowest values were discarded and the mean of the other 13 fields was calculated, and values were expressed as the percentage of C5b-9 deposits induced by a pool of sera from 10 healthy controls run in parallel (reference 100%). Dashed lines indicate upper and lower limit of normal range. (B) HMEC-1 were activated with ADP and exposed for 2 h to serum (diluted 1:2 with test medium, HBSS with 0.5% BSA) from the patient or with a control serum pool. Perfusion of heparinized whole blood (heparin 10 U/ml) from an healthy subject (added with the fluorescent dye mepacrine 10 microM, to label platelets) was then performed in a thermostatic flow chamber (37 C) in which one surface of the perfusion channel was a glass slide seeded with a monolayer of endothelial cells at a constant flow rate of 1500 sec-1 (60 dynes/cm2). After 3 min, perfusion was stopped, and the slide with the endothelial cell monolayer was dehydrated and fixed in acetone for 20 min. Slides were examined under confocal inverted laser microscopy. Fifteen fields for each slide were systematically digitized along the surface and the area covered by thrombi was quantified by Image J (NIH, Bethesda, MD), and expressed as pixel2 per field analyzed. For each sample the mean of 15 fields (excluding the lowest and the highest values) was calculated. Dashed lines indicated the area covered by thrombi of control serum pool +- SE. Data are reported as means +- SE.  P<0.0001,   P<0.001 versus control serum pool; ^P<0.0001 versus day 0; *P<0.0001 versus day 1; #P<0.0001, ##P<0.001 versus day 7. Statistical analysis: ANOVA.
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CT imaging 13 days after the infusion showed a marked decrease of the consolidation areas with unmodified reticular abnormalities and bronchiectasis. Consistently, ex vivo serum induced C5b-9 deposition and thrombi formation on HMEC-1 line, after an initial acute increase the day after antibody infusion, progressively decreased, and eventually normalized ( Figures 3A, B , respectively).
D-Dimer levels in patient plasma samples collected before antibody infusion (Day 0) and 1, 7, 14, and 21 days thereafter. Dashed line represents the upper normal reference value.
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Similarly, plasma fibrinogen concentration, vWF antigen and activity, and D-Dimer levels ( Figure 4 ), which were remarkably increased before antibody infusion, slightly increased at post infusion day 1 and then progressively decreased on subsequent visits. In particular, D-dimer levels decreased to normal range at post infusion days 14 and 21 ( Figure 4 ).
PMC8278474_02
Levels of anti-SARS-CoV-2 N IgG antibodies in the concentrate antibody solution infused to the patient, in antibody donor serum and in recipient sera collected before antibody infusion (day 0) and 7, 14, and 22 days thereafter. The dashed line indicates the threshold for antibody positivity.
fimmu-12-711915-g002.jpg
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Seven hundred milliliter of a saline solution of anti-SARS-CoV-2 N IgG (Index 8.8,  Figure 2 ) were obtained (after filter washing at the end of the DFPP session) from a 61-year-old male ABO competent adult donor who, according to the clinical and laboratory criteria issued by Consiglio Superiore di Sanita on February 28, 2020, had definitely recovered from COVID-19. The donor was found to have an adequate titer of anti-SARS-CoV-2 S1/S2 (84 UA/ml) and anti-N IgG (7.74 index,  Figure 2 ). SARS-CoV-2 S1/S2 IgG test (DiaSorin) and subsequently confirmed by chemiluminescence microparticle immunoassay (CMIA) Alinity SARS-CoV-2 IgG (Abbott) detection of anti-nucleocapsid antigen (N) circulating IgG. Serum levels of anti-SARS-CoV-2 N IgG antibodies, which were almost undetectable before antibody infusion, sharply increased at post-infusion day 1 and then slowly and progressively declined at post-infusion days 7, 14, and 21 ( Figure 2 ). However, following the infusion antibody titer largely exceeded the upper limit for test positivity (index 1.2) up to day 21 ( Figure 2 ).
Patient serum-induced ex vivo C5b-9 deposition (A) and thrombi formation (B) on cultured Human Microvascular Endothelial Cells (HMEC-1) line immediately before antibody infusion (Day 0), and 1, 7, 14, and 21 days thereafter. (A) HMEC-1 were incubated for 2 h with serum (diluted 1:2 with test medium, HBSS with 0.5% BSA) from the patient or with a control serum pool. At the end of incubation, cells were washed, fixed, and stained with rabbit anti-human complement C5b-9 complex antibody followed by FITC-conjugated secondary antibody. Fluorescence microscopy was used to view the fluorescent staining on endothelial cell surface, and the HMEC-1 area covered by C5b-9 staining was calculated by automatic edge detection (Image J software) in 15 high power fields. For each sample, the highest and the lowest values were discarded and the mean of the other 13 fields was calculated, and values were expressed as the percentage of C5b-9 deposits induced by a pool of sera from 10 healthy controls run in parallel (reference 100%). Dashed lines indicate upper and lower limit of normal range. (B) HMEC-1 were activated with ADP and exposed for 2 h to serum (diluted 1:2 with test medium, HBSS with 0.5% BSA) from the patient or with a control serum pool. Perfusion of heparinized whole blood (heparin 10 U/ml) from an healthy subject (added with the fluorescent dye mepacrine 10 microM, to label platelets) was then performed in a thermostatic flow chamber (37 C) in which one surface of the perfusion channel was a glass slide seeded with a monolayer of endothelial cells at a constant flow rate of 1500 sec-1 (60 dynes/cm2). After 3 min, perfusion was stopped, and the slide with the endothelial cell monolayer was dehydrated and fixed in acetone for 20 min. Slides were examined under confocal inverted laser microscopy. Fifteen fields for each slide were systematically digitized along the surface and the area covered by thrombi was quantified by Image J (NIH, Bethesda, MD), and expressed as pixel2 per field analyzed. For each sample the mean of 15 fields (excluding the lowest and the highest values) was calculated. Dashed lines indicated the area covered by thrombi of control serum pool +- SE. Data are reported as means +- SE.  P<0.0001,   P<0.001 versus control serum pool; ^P<0.0001 versus day 0; *P<0.0001 versus day 1; #P<0.0001, ##P<0.001 versus day 7. Statistical analysis: ANOVA.
fimmu-12-711915-g003.jpg
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CT imaging 13 days after the infusion showed a marked decrease of the consolidation areas with unmodified reticular abnormalities and bronchiectasis. Consistently, ex vivo serum induced C5b-9 deposition and thrombi formation on HMEC-1 line, after an initial acute increase the day after antibody infusion, progressively decreased, and eventually normalized ( Figures 3A, B , respectively).
D-Dimer levels in patient plasma samples collected before antibody infusion (Day 0) and 1, 7, 14, and 21 days thereafter. Dashed line represents the upper normal reference value.
fimmu-12-711915-g004.jpg
PMC8278474_02_fimmu-12-711915-g004.jpg
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Similarly, plasma fibrinogen concentration, vWF antigen and activity, and D-Dimer levels ( Figure 4 ), which were remarkably increased before antibody infusion, slightly increased at post infusion day 1 and then progressively decreased on subsequent visits. In particular, D-dimer levels decreased to normal range at post infusion days 14 and 21 ( Figure 4 ).