Despite vaccination (Moderna, booster 6 months earlier), a 34-year-old patient affected by B-lymphoblastic leukemia t(9;22)(q34;q11.2) BCR-ABL1 developed Omicron BA.5 infection while waiting for her scheduled transplant from a matched unrelated donor (MUD). She was referred to our center for allo-HSCT after treatment with Imatinib, which was withdrawn after one week for intolerance in favor of Dasatinib obtaining a first hematological complete remission with minimal residual disease (MRD) positivity. Subsequently, the patient received Ponatinib due to increased MRD levels according to GIMEMA LAL2620 (NCT04475731), but the disease relapsed. After achieving second complete morphological remission with detectable MRD after two cycles of Inotuzumab ozogamicin (IO), an allo-HSCT was scheduled.  Figure 1  displays the Next-Generation Sequencing (NGS) analysis of the virus.
The patient received prompt preventive antiviral therapy with nirmatrelvir/ritonavir, obtaining fever resolution within 72 hours and a consistent viral load reduction at nasopharyngeal swabs. Twenty-three days after COVID-19 diagnosis, based on increasing MRD values in the context of high-risk disease refractory to third generation tyrosine kinase inhibitor and clinical resolution of SARS-CoV-2 infection, it was decided not to delay further allo-HSCT. During conditioning chemotherapy [fludarabin, 12 Gy total body irradiation (TBI)], nasopharyngeal viral load increased (virus confirmed viable in cell culture), while the patient remained asymptomatic. Two days before PBSC reinfusion in July 2022, tixagevimab/cilgavimab 300/300 mg and a 3-day course of remdesivir were administered. The donor was a female 10/10 HLA-MUD, vaccinated for SARS-CoV-2. The graft-versus host disease (GVHD) prophylaxis was based on post-transplant cyclophosphamide (PT-Cy), mycophenolate mofetil, and cyclosporine A (CsA). Neutrophil engraftment was reached at +20 days after the transplant.
The pre-engraftment phase was characterized by uncomplicated febrile neutropenia without documented infections. Despite trying to minimize the risk factors for veno-occlusive disease (VOD) in the context of IO exposure and a TBI-based myeloablative-preparing regimen, administering ursodeoxycholic acid prophylaxis and using CsA rather than sirolimus as GVHD prophylaxis, VOD occurred at day +13 (thinning of hepatic veins, thickening of gallbladder, ascites, bilirubin peak 2.3 mg/dL, Fibroscan peak 75kPa), requiring defibrotide treatment to obtain a slow but complete resolution of this complication.
The post-engraftment phase was characterized by mild COVID-19 at day +23 (cough, rhino-conjunctivitis, fever). A spontaneous symptom resolution was observed, enabling the patient to achieve a definitive viral clearance at day +28 after transplant. At day +32, the patient experienced grade I acute GVHD (skin grade II) treated with methylprednisolone 0.5 mg/kg/daily and three cycles of weekly extracorporeal photo-apheresis as a steroid-sparing approach due to concomitant SARS-2-CoV infection, without side-effects. At day +40 after transplant with a full donor chimerism of myeloid and T-cells and in complete remission with negative MRD, the patient was discharged home. Then, a steroid dose increase was required to obtain GVHD remission, without further complications or adverse events during follow-up until day +180. Bone marrow evaluation performed at six months confirmed complete hematological remission with negative MRD, without requiring additional treatments.
  Figure 2  summarizes the comprehensive clinical course of SARS-CoV-2 nasopharyngeal viral load trends by PCR cycles, C-reactive protein, D-dimer values, COVID-19 treatments in pre and post-transplant phases, veno-occlusive disease, and acute GVHD treatments.
SARS-CoV-2-specific cellular and humoral responses were also analyzed in detail and detailed methods are reported in the Supplementary Materials of this case study.
IgGs against the SARS-CoV-2 RBD were present before transplant although the omicron RBD antibody binding was 7 and 4-fold lower compared with that of the Wuhan-Hu-1 and delta RBDs, respectively. Post tixagevimab-cilgavimab administration, anti-RBD IgG levels against Wuhan and delta increased and followed a similar kinetic over time, while an omicron IgG rise lagged until day +30, which is consistent with the reduced binding by tixagevimab-cilgavimab to this VOC ( Figure 3A ). Binding IgGs against the SARS-CoV-2 nucleocapsid protein were initially negative and developed by day +37 after the positive swab.
Neutralizing Ab (NAb) against delta and omicron VOC were at low titers (1/82 and 1/66 respectively) twenty-one days after the positive swab, a time point which was, however, two days after chemotherapy ( Figure 3B ). NAb against both VOCs showed increased titers when tested at twelve days after administration of tixagevimab-cilgavimab and had similar kinetics thereafter. Overall, the neutralizing response to omicron was persistently lower than that of delta (fold range: 2-90). None of the serum samples neutralized the LV-Luc/VSV.G pseudo-viral vector, indicating that the detected neutralizing responses were specific for SARS-CoV2 (data not shown).
Both polyclonal ( Figure 3C ) and SARS-CoV-2 specific T-cell reconstitution ( Figure 3D ) were longitudinally evaluated before (-4 days) and at day +30, +60 and +90 after HSCT. Before the transplant, the patient showed a limited but detectable level of specific responses toward the Spike (6 SFC/400'000 PBMC) and the Nucleocapsid protein (5 SFC/400'000 PBMC). Early after transplant (+30 days), the frequency of both polyclonal and SARS-CoV-2 specific T-cell responses were below the limit of detection of the assay due to the low CD3+ T-cell counts (59 CD3+ T cells/microl, 48 CD4+ T cells/microl, and 11 CD8+ T cells/microl). At the following time points, the absolute counts of CD3+ T cells increased (379 and 353 CD3+ T cells/microl at day +60 and +90, respectively). Accordingly, the polyclonal T-cell response rose to 1'664 SFC/400'000 PBMC at day +90 after HSCT. However, the specific response to the Nucleocapsid protein remained negative after transplant, and the frequency of Spike-specific T-cells was assessed to be at very low levels (3 SFC/400'000 PBMC at both days +60 and +90). A progressive decrease of the viral load was observed within a few weeks after transplant and the molecular SARS-CoV-2 nasopharyngeal swab test was negative by day +28 after transplant, before the reconstitution of an adequate number of T-cells that could elicit a specific response.