PMC7732473_01
PMC7732473_02
Immune reconstitution after gene therapy (GT). (A) Engraftment of gene corrected cells expressed as VCN/genome in sorted subpopulations from peripheral blood (PB), measured by Real Time-PCR during follow-up after GT. VCN, Vector Copy Number. (B) WASP expression (% of WASP+ cells) by flow cytometry in PB cell subpopulations. WASP, WAS protein. (C) Peripheral blood cell counts at different time points before GT and during follow-up. Bu, Busulfan; Flu, fludarabine. *The 6 month-follow up visit was performed between 6.4 and 6.9 months after GT, after second negative swab for SARS-CoV2.
fimmu-11-603428-g001.jpg
PMC7732473_05_fimmu-11-603428-g001.jpg
f1
After complete resolution of SARS-CoV-2 infection, molecular and immunological tests showed persistence of engraftment of gene corrected cells, with marked rise in CD8+ transduced cells, increase in WASP-expressing cells and stable lymphocyte count ( Figure 1 ). IL-6 increased transiently during acute SARS-CoV-2 infection and then returned to normal values ( Figure 1A ).
PMC7732473_03
Immune response to SARS-CoV-2 infection. (A) Lymphocyte subpopulations counts and inflammatory marker levels during SARS-CoV-2 infection. SARS-CoV-2 E gene real time (RT)-PCR cycle threshold (Ct) on nasopharyngeal (NP) swabs are reported, when available. +, positive; +/-, weakly positive; -, negative. HCQ, hydroxychloroquine; LPV/r, lopinavir/ritonavir. CVC, central venous catheter. Units of measure of data reported on y axis are specified in the legend on the right. (B, C) Anti-SARS-CoV-2 and anti-OC43/HKU1 Coronaviruses antibody serum level in our patient (B), in three children and in patient's mum (black stars) after SARS-CoV-2 infection (C). (D) 50% neutralization (IC50) (expressed as reciprocal of serum dilution) of SARS-CoV-2 with the serum of our patient and of another child after SARS-CoV-2 infection. The first dilution tested was 1/40.
fimmu-11-603428-g002.jpg
PMC7732473_05_fimmu-11-603428-g002.jpg
f2
Spike proteins (S1S2) were positive by day 6 after first positive swab and at day 61 ( Figure 2B ), reaching levels comparable to those of children who experienced SARS-CoV2 infection (n=3) and higher than the mother ( Figure 2C ). Serum IgA and IgM to RBD were also detected in the patient. Notably, at all time points the patient had a stable positive signal for antibodies against two coronaviruses associated with common cold (HKU1 and OC43) ( Figure 2B ). Since the patient was still on monthly IVIg supplementation, we tested eight children receiving Ig supplementation, all with negative SARS-CoV-2 swabs, and followed at our Unit in the same period. Remarkably, the patient's serum also showed a neutralizing capacity to SARS-CoV2 using a Spike-pseudotyped lentiviral vector in a VERO E6 cell assay ( Figure 2D ) (manuscript in preparation).
PMC7732473_04
PMC7732473_05
Immune reconstitution after gene therapy (GT). (A) Engraftment of gene corrected cells expressed as VCN/genome in sorted subpopulations from peripheral blood (PB), measured by Real Time-PCR during follow-up after GT. VCN, Vector Copy Number. (B) WASP expression (% of WASP+ cells) by flow cytometry in PB cell subpopulations. WASP, WAS protein. (C) Peripheral blood cell counts at different time points before GT and during follow-up. Bu, Busulfan; Flu, fludarabine. *The 6 month-follow up visit was performed between 6.4 and 6.9 months after GT, after second negative swab for SARS-CoV2.
fimmu-11-603428-g001.jpg
PMC7732473_05_fimmu-11-603428-g001.jpg
f1
After complete resolution of SARS-CoV-2 infection, molecular and immunological tests showed persistence of engraftment of gene corrected cells, with marked rise in CD8+ transduced cells, increase in WASP-expressing cells and stable lymphocyte count ( Figure 1 ). IL-6 increased transiently during acute SARS-CoV-2 infection and then returned to normal values ( Figure 1A ).
Immune response to SARS-CoV-2 infection. (A) Lymphocyte subpopulations counts and inflammatory marker levels during SARS-CoV-2 infection. SARS-CoV-2 E gene real time (RT)-PCR cycle threshold (Ct) on nasopharyngeal (NP) swabs are reported, when available. +, positive; +/-, weakly positive; -, negative. HCQ, hydroxychloroquine; LPV/r, lopinavir/ritonavir. CVC, central venous catheter. Units of measure of data reported on y axis are specified in the legend on the right. (B, C) Anti-SARS-CoV-2 and anti-OC43/HKU1 Coronaviruses antibody serum level in our patient (B), in three children and in patient's mum (black stars) after SARS-CoV-2 infection (C). (D) 50% neutralization (IC50) (expressed as reciprocal of serum dilution) of SARS-CoV-2 with the serum of our patient and of another child after SARS-CoV-2 infection. The first dilution tested was 1/40.
fimmu-11-603428-g002.jpg
PMC7732473_05_fimmu-11-603428-g002.jpg
f2
Spike proteins (S1S2) were positive by day 6 after first positive swab and at day 61 ( Figure 2B ), reaching levels comparable to those of children who experienced SARS-CoV2 infection (n=3) and higher than the mother ( Figure 2C ). Serum IgA and IgM to RBD were also detected in the patient. Notably, at all time points the patient had a stable positive signal for antibodies against two coronaviruses associated with common cold (HKU1 and OC43) ( Figure 2B ). Since the patient was still on monthly IVIg supplementation, we tested eight children receiving Ig supplementation, all with negative SARS-CoV-2 swabs, and followed at our Unit in the same period. Remarkably, the patient's serum also showed a neutralizing capacity to SARS-CoV2 using a Spike-pseudotyped lentiviral vector in a VERO E6 cell assay ( Figure 2D ) (manuscript in preparation).