Effective virus-specific T-cell therapy for high-risk SARS-CoV-2 infections in hematopoietic stem cell transplant recipients: initial case studies and literature review

The COVID-19 pandemic has exacerbated mortality rates among immunocompromised patients, accentuating the need for novel, targeted therapies. Transplant recipients, with their inherent immune vulnerabilities, represent a subgroup at significantly heightened risk. Current conventional therapies often demonstrate limited effectiveness in these patients, calling for innovative treatment approaches. In immunocompromised transplant recipients, several viral infections have been successfully treated by adoptive transfer of virus-specific T-cells (VST). This paper details the successful application of SARS-CoV-2-specific memory T-cell therapy, produced by an interferon-γ cytokine capture system (CliniMACS® Prodigy device), in three stem cell transplant recipients diagnosed with COVID-19 (case 1: alpha variant, cases 2 and 3: delta variants). These patients exhibited persistent SARS-CoV-2 PCR positivity accompanied by bilateral pulmonary infiltrates and demonstrated only partial response to standard treatments. Remarkably, all three patients recovered and achieved viral clearance within 3 to 9 weeks post-VST treatment. Laboratory follow-up investigations identified an increase in SARS-CoV-2-specific T-cells in two of the cases. A robust anti-SARS-CoV-2 S (S1/S2) IgG serological response was also recorded, albeit with varying titers. The induction of memory T-cells within the CD4 + compartment was confirmed, and previously elevated interleukin-6 (IL-6) and IL-8 levels normalized post-VST therapy. The treatment was well tolerated with no observed adverse effects. While the need for specialized equipment and costs associated with VST therapy present potential challenges, the limited treatment options currently available for COVID-19 within the allogeneic stem cell transplant population, combined with the risk posed by emerging SARS-CoV-2 mutations, underscore the potential of VST therapy in future clinical practice. This therapeutic approach may be particularly beneficial for elderly patients with multiple comorbidities and weakened immune systems. Supplementary information The online version contains supplementary material available at 10.1007/s11357-023-00858-7.

In immunocompromised transplant recipients, several viral infections have been successfully treated by adoptive transfer of virus-specific T-cells (VST).This paper details the successful application of SARS-CoV-2-specific memory T-cell therapy, produced by an interferon-γ cytokine capture system (Clini-MACS® Prodigy device), in three stem cell transplant recipients diagnosed with COVID-19 (case 1: alpha variant, cases 2 and 3: delta variants).These patients exhibited persistent SARS-CoV-2 PCR positivity accompanied by bilateral pulmonary infiltrates and demonstrated only partial response to standard treatments.Remarkably, all three patients recovered and achieved viral clearance within 3 to 9 weeks post-VST treatment.Laboratory follow-up investigations identified an increase in SARS-CoV-2-specific

Introduction
As of May 2023, the World Health Organization (WHO) reported over 765 million confirmed cases and more than 6.9 million deaths globally due to COVID-19 [1].Since the pandemic's inception, the severity of COVID-19 infections among elderly, immunocompromised, and co-morbid individuals, including those undergoing immunosuppressive therapy, solid organ transplantation (SOT), or hematopoietic stem cell transplantation (HSCT), has been of significant concern [2][3][4].Age-related immunosenescence may contribute significantly to the severity of COVID-19 infection and viral persistence [5].A syndrome akin to Long COVID-19, characterized by protracted or recurrent symptoms, repeated viremia, and lung complications, is prevalent in SOT/HSCT recipients and can be accompanied by latent myocardial, renal, and neurological conditions [5].
The SARS-CoV-2 pandemic has presented a considerable challenge for patients with malignant hematological diseases and those undergoing HSCT [6,7].A survey by the Center for International Blood and Marrow Transplant Research (CIBMTR) reported a 30-day COVID-19 mortality rate exceeding 30% for both transplant modalities [7].European Society for Blood and Marrow Transplantation (EBMT) data also indicated substantial mortality rates [8].Follow-up studies concluded that COVID-19 poses a high risk to HSCT patients, especially those undergoing allogenic-HSCT and autologous HSCT within 6 months, necessitating special monitoring [2].While initial reports indicated higher in-hospital mortality among SOT recipients, subsequent studies revealed a survival rate comparable to the general population.However, SOT recipients over 60 demonstrated a higher mortality rate and were more likely to receive treatments such as remdesivir, convalescent fresh frozen plasma (FFP), and biological therapy [9,10].
Anti-viral and biological treatment approaches often remain ineffective; therefore, alternative therapeutic options need to be considered [11][12][13][14].In addition to life threatening outcomes, a persistent SARS-CoV-2 infection in immunocompromised hosts also carries the risk of giving rise to series of mutations in the viral genome [15][16][17][18].Reports of prolonged viral shedding and recurrent relapses in SOT recipients have raised concerns about the emergence of multimutation variants, possibly leading to immune escape variants [9,19].Drawing from experiences with other viral reactivations, the adoptive transfer of T-cell therapy was proposed to combat and clear the SARS-CoV-2 virus [20,21].To date, adoptive transfer of virus-specific T-cells (VST) after allogeneic HSCT or SOT has shown promise in treating several resistant or persistent viral reactivations and diseases [22].The first successful use was reported against cytomegalovirus (CMV) reactivation/disease, Epstein-Barr virus (EBV)-associated lymphoma, resulting in complete Vol.: (0123456789) remission in a significant proportion of patients [23,24].Presently, VST products for clinical use can be manufactured through ex vivo expansion or direct selection, although the former tends to be complex, time-consuming, and costly [25].Direct selectionproduced VSTs provide fresh insights into therapeutic approaches for refractory viral infections, with a major advantage being their rapid production within 12-24 h [26].Over the past decade, an interferon-γ (IFN-γ) cytokine capture system (CCS), used via a fully automated and closed technique on the Clini-MACS® Prodigy device, has been employed to treat viral infections in allogeneic HSCT settings [26].
At our center, we have successfully utilized VST treatments for viral reactivations/diseases in pediatric HSCT recipients [27,28].We now present the successful application of SARS-CoV-2 VST therapy using the IFN-γ CCS in the first three HSCT recipients, along with a discussion on key laboratory parameters.

SARS-CoV-2 VST recipients
The inclusion criteria for SARS-CoV-2 VST therapy were the following: severe or critical COVID-19 in immunocompromised or HSCT recipients with specific indications for VST treatment: (i) not responding to at least 2 anti-COVID-19 therapies (anti-viral and/or biological treatment); (ii) showing persistent pulmonary infiltration, recurrent symptoms, or persistent PCR positivity (peripheral blood or nasopharyngeal swab); (iii) with at least 1 or 2 human leukocyte antigen (HLA) allele matches between recipient and donor (based on HLA-A, B, C, DR).Exclusion criteria for VST treatment were as follows: (1) absence of HLA match, (2) previous anaphylactic reaction to a blood product, (3) ongoing treatment of methylprednisolone or dexamethasone, and (4) patient unwillingness.The study was supported by the Institutional Board and approved by the Scientific and Research Ethics Committee of the Hungarian National Medical Scientific Council (ETT-TUKEB IV/2743-1/2021/ EKU).For prospective data collection and analysis, all patients signed a separate informed consent form beside the EBMT/CIBMTR consent forms.

SARS-CoV-2 VST donors
The VST donors were selected from the Hungarian National Blood Transfusion Service (OVSZ) volunteer stem cell donor system, based on their HLA-A, -B, and -DR antigen matches and COVID-19 status (either convalescent and/or vaccinated).Donors were screened by flow cytometry with SARS-CoV-2 peptide pool kit (for details, see flow cytometry methods).Donors also signed an informed consent form for participating in the procedure.

Case 1
A 50-year-old male patient with intermediate risk acute myeloid leukemia (AML) in measurable residual disease (MRD) negative, first complete remission (CR) underwent allogeneic-HSCT from a 10/12 (2 HLA-DPB1 antigen mismatched) matched unrelated donor with granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell (PBSC) product (Table 1).At day + 63, he developed grade 2 acute skin GVHD.On day + 70, the patient experienced sore throat without any systemic symptoms.The nasopharyngeal swab showed SARS-CoV-2 PCR positivity.The chest CT-scan was normal.The patient remained on methylprednisolone and ruxolitinib combination, and remdesivir was added.SARS-CoV-2 RNAemia was detected on day + 84; therefore, he received 3 × 2 units of COVID-19 convalescent fresh frozen plasma.Because of radiological progression, the patient was switched to baricitinib and remdesivir from day + 134.During persistent COVID-19 and in addition to poor graft function complicated by neutropenic fevers, fungal infection and multiple CMV reactivations required broad-spectrum antibiotic, posaconazol, amphotericin-B, and foscarnet treatments that were required during the hospitalization.After more than 5 months, the nasopharyngeal swab and blood PCR positivity persisted, and CT-scans showed gradual progression of bilateral ground glass opacities, consolidations, reticular, and inter-septal thickening accompanied by pleural fluid accumulation corresponding to moderate COVID-19 pneumonia.Based on the donor screening, a 4/6 HLA-matched convalescent male donor was identified.The patient received 2 doses of 5 × 10 3 /kg SARS-CoV-2 VST from the same donor on days + 222 and + 229 post-transplant.

Case 2
A 31-year-old male presented with BCR:ABL1 positive acute lymphoid leukemia (Ph + ALL).In first CR with molecular response 5.0, he underwent allogeneic-HSCT from HLA-identical male sibling donor with G-CSF-mobilized PBSC product following with total-body irradiation (TBI) and etoposide (Table 1).On day + 150, late-onset acute grade 2 GVHD with skin and gut involvement developed.He commenced treatment with methylprednisolone and ruxolitinib, resulting in a prompt and complete resolution.On day + 177, while asymptomatic, a nasopharyngeal swab screening sample confirmed PCR positivity for SARS-CoV-2.Being at high risk, the patient received favipiravir and bamlanivimab therapy in the outpatient clinic.Eight days later, a fever developed.Remdesivir and dexamethasone treatment and 2 × 2 units of COVID-19 convalescent fresh frozen plasma were administered at the Infectious Disease Department.Prior to SARS-CoV-2 VST treatment, the chest CT scan showed bilateral interstitial pneumonia.After more than 6 weeks of persistent SARS-CoV-2 PCR positivity, the patient was invited to participate in the SARS-CoV-2 VST program.Based on the donor screening tests, a young female volunteer vaccinated twice with Pfizer-BioNTech mRNA vaccine turned out to be a 4/6 HLA-matched.On days + 225 and + 240, the patient received two doses of 5 × 10 3 / kg SARS-CoV-2 VST from the same donor.Two weeks after COVID-19 VST treatment, complete regression of pneumonia was detected by Chest CT.

Case 3
A 40-year-old male patient diagnosed with peripheral T-cell lymphoma underwent autologous-HSCT following a conditioning with thiotepa, cytosinearabinoside, etoposide, and melphalan (TEAM) in MRD negative first CR (Table 1).Five months after autologous transplant, the T-cell lymphoma relapsed, and SARS-CoV-2 infection was concurrently revealed with bilateral interstitial pneumonitis on the CT-scan.The patient received dexamethasone and remdesivir treatment, which resulted in marked regression of infiltrates on CT.However, nasopharyngeal swab PCR positivity persisted, and residual pneumonia did not show further regression.Based on donor screening tests, a young 4/6 HLA-matched male convalescent donor with 2 × Gam-COVID-Vac (Sputnyik V) vaccination was found to be suitable.The patient received 2 doses of 5 × 10 4 /kg SARS-CoV-2 VST from the same donor 1 week apart on days + 218 and + 225 after autologous-HSCT.

Characterization of the screened VST donors
Altogether, 17 donors with appropriate HLA matching and COVID-19 status for transplant recipients were investigated (3 convalescent, 12 vaccinated, and 2 combined).Although the number of cases is

Clinical course after VST administration
Following VST therapy, all three patients recovered from COVID-19 and achieved viral clearance (Table 1).No short-term side effects were observed after treatment with SARS-CoV-2 VST, including GVHD or cytokine release syndrome (CRS).However, after a few months of complete recovery from COVID-19, GVHD relapse was observed in both allogeneic transplant recipients, indicating recovery of immune functions.In terms of long-term outcome (11 months of follow-up), 2 out of 3 patients are alive.In case 1, the patient's condition gradually improved, and CT abnormalities showed moderate regression.On day + 241, due to persistent poor graft function with 100% donor cell chimerism, he received a CD34 + cell booster with 1 × 10 6 /kg CD3 + T-cells from his original unrelated donor resulting improving condition and blood counts.On day + 261, the patient was discharged from hospital.The patient was checked at the outpatient clinic for 1.5 months with a gradually improving condition and blood counts.On day + 302, he developed fever and dyspnea requiring re-admission.The chest CT-scan showed bilateral, nodular, and multifocal peribronchial infiltrations.The SARS-CoV-2 PCR for nasopharyngeal swab and blood samples remained negative.Five days after the admission, the patient developed respiratory failure, requiring intubation and mechanical ventilation.Persistent fever, severe pancytopenia, increased IL-6 (189.5 pg/ml) and fibrinogen (6.4 g/l), and extremely high ferritin levels (23,960 ng/ml) developed.Causative etiopathogens could not be verified during microbiological assessment.Laboratory and clinical parameters corresponded to chronic GVHDinduced secondary hemophagocytic-lymphohistiocytosis (HLH) associated idiopathic pneumonia syndrome (IPS) with acute respiratory distress syndrome (ARDS).A combination of high-dose methylprednisolone, ruxolitinib, tocilizumab, and etanercept were ineffective.Despite ventilatory and hemodynamic supports, the patient's clinical condition gradually deteriorated, and he died 15 days after admission.Autopsy was not performed.In case 2, the patient Vol:.(1234567890) developed chronic extensive GVHD treated with ruxolitinib and extracorporeal photopheresis (ECP) resulting in good clinical response.Two weeks after COVID-19 VST treatment, complete regression of pneumonia was detected by chest CT.Eleven months after VST therapy, the patient's condition is excellent, free from SARS-CoV-2, with good blood counts and his underlying disease in remission.In case 3, VST treatment resulted in complete regression of bilateral pneumonia.Eleven months after VST therapy, the patient's condition is excellent.Peripheral T-cell lymphoma activity was detected on PET/CT-scan indicating a combination treatment with azacytidine, dexamethasone, and romidepsine.Furthermore, he developed myelodysplastic syndrome with multilineage dysplasia, therefore, becoming a candidate for an allogeneic-HSCT from his HLA-identical sibling donor.

Characterization of peripheral blood lymphoid cell subpopulations by flow cytometry
The CD3 + , CD3 + CD4 + , or CD3 + CD8 + T-cell ratios showed no marked change after VST therapy (Fig. 1A-C, Supplementary Table 3).VST treatment did not cause measurable changes in the major T-cell subsets.In all patients, a decrease in B cell ratio was observed at week 3 after VST administration, followed by a slow, gradual recovery in the subsequent 2 months (Fig. 1D).The increase in B cells 2 months after recovery from COVID-19 infection possibly indicates immune system regeneration.The proportion of B cells following SARS-CoV-2 virus clearance, however, showed an upward trend compared to previous values, albeit to varying degrees.In the case of the 2 allogeneic transplant recipients, the return of GVHD after COVID-19 recovery can also be considered as an indirect sign.In case 1, unlike other patients, TCRγδ + T-cell growth indicating the dominance of the innate immune system was detected during the course of COVID-19.Following VST treatment, gradual recovery and dominance of TCRαβ + T-cells representing the adaptive immune system was observed in two months post-VST (Fig. 1E).In the remaining 2 patients, a dominance of TCRαβ + T-cells was observed.At week 3 following VST treatment, we experienced a nadir in the naive CD4 + CD45RA + T-cell counts (Fig. 1G).After the recovery from COVID-19, a gradual increase was detected.CD4 + CD45RO + memory T-cells showed expansion in all patients by week 3, and later, their ratio stabilized (Fig. 1H).CD8 + CD45RO + compartments did not show any characteristic fluctuation after VST therapy, and after 2 months, their proportion stabilized between 30 and 40% (Fig. 1J).
Monitoring SARS-CoV-2 RNA by PCR in blood and nasopharyngeal swab samples SARS-CoV-2 PCR negativity could be achieved in all three patients by 3-9 weeks after VST treatment.PCR negativity, indicating achievement of viral clearance, was defined as a Ct count above 40.Although the Ct number is a semi-quantitative method, it provides good information on the dynamics of the process.In case 1, both nasopharyngeal swab and peripheral blood specimens showed persistent PCR positivity for more than 5 months before VST therapy.By week 3 following the first dose of VST, blood samples became PCR negative.Nasopharyngeal clearance occurred by 9 weeks after VST therapy.In case 2, peripheral blood PCR positivity cleared by week 1 and nasopharyngeal swabs by week 3 after VST therapy.In case 3, blood SARS-CoV-2 PCR become negative after the antiviral treatment, but nasopharyngeal swab positivity persisted.The viral RNA clearance from nasopharyngeal swab occurred at week 4 post VST treatment (Supplementary Table 4).

Monitoring SARS-CoV-2-specific antibody titers
In case 1, antibody response could not be detected during screening despite a long-lasting COVID-19 (Fig. 3A).Definite antibody responses emerged following VST treatment.SARS-CoV-2 S1/S2-specific IgG was first to appear at week 1 after the VST infusion, and the titer continued to rise during the following weeks.At week 3, the titer was 53.5 AU/ml, and a measurable neutralizing antibody titer was detected.The anti-SARS CoV-2 S1/S2 IgG titer was found to be 382 AU/ml in week 5 and > 400 AU/ml in week 9. Case 2 behaved differently: anti-SARS-CoV-2 S1/S2 IgG was present even in a neutralizing antibody titer at screening, which increased following VST therapy followed by a gradual decrease (Fig. 3B).However, SARS-CoV-2 NP IgG did not appear.In case 3, all SARS-CoV-2 antibody tests were negative at the time of screening (Fig. 3C).As a result of VST treatment, the anti-SARS-CoV-2 S1/S2 IgG became positive but never reached the neutralizing titer.Furthermore, anti-SARS-CoV-2 NP IgG also did not appear.

Discussion
To our knowledge, this is the pioneering case series to report successful implementation of SARS-CoV-2 VST therapy, employing direct isolation through an IFN-γ CCS with the CliniMACS® Prodigy System, in adult stem cell transplant recipients diagnosed with COVID-19.Post-VST treatment, all three patients displayed regression of pulmonary infiltrates and achieved viral clearance, as demonstrated by SARS-CoV-2 PCR negativity in blood and nasopharyngeal swab samples within a span of 3-9 weeks.
Both allogeneic patients had acute GVHD prior to COVID-19 infection, which subsided due to persisting infection, and even immunosuppression treatment could be discontinued.Furthermore, after the recovery of COVID-19 infection, GVHD returned, then already in the form of a chronic process.Of course, the exact mechanism of this process is unknown, but based on recent observations, it may indeed be related to the spread of immune exhaustion, immunosenescence and SARS-CoV-2 induced senescence phenomenon.The resurgence of chronic GVHD was interpreted as an indication of the recuperation of the donor immune system post-COVID-19 infection, which could be an outcome of the CD34 + booster administered in case 1.In addition, these observations may raise the hypothesis that SARS-CoV-2-induced senescence phenomenon may increase the risk of developing many pathological process in the medium to long-term.SARS-CoV-2-specific memory T-cells were detected in both allogeneic transplant recipients.Notably, by the 5th week of VST treatment in the first case, the ratio of CD4 + IFNγ + SARS-CoV-2-specific T-cells even exceeded the numbers observed in 17 donors from our current study, as well as those reported by Ferreras et al. [31].In case 3, no CD4 + / CD8 + IFNγ + SARS-CoV-2-specific T-cells appeared despite VST treatment, which could be explained by the relapse of T-cell lymphoma, causing a long-term immunosuppressive state.When using VST products, cross-reactivity with other viruses cannot be ruled out, but the significance of the above is difficult to determine [32].Clearly, SARS-CoV-2-specific humoral responses showed strong differences in all three patients.Similar humoral immune responses observed in convasecent subjects prior to the appearance of vaccines include SARS-CoV-2 anti-spike IgG levels with variable titers, often below neutralizing levels, and highly diverse anti-nucleocapsid IgG (often negative) responses.In contrast, with the proliferation of vaccines, there was often a high spike IgG response and a distinctly elevated neuralizing titer without nucleocapsid IgG.Accordingly, since patients received an adoptive T-cell transfer therapy, the humoral responses to the patch correspond to the pattern of very wide antibody responses observed among convalescent subjects.In the first case, both SARS-CoV-2 spike IgG, IgA, neutralizing titer, and NP IgG demonstrated a clear humoral response.In the second case, the SARS-CoV-2 spike IgG and neutralizing titer continued to rise under the influence of the VST, but NP IgG did not appear.Case 3, which took place in the immunosuppressive milieu of active an T-cell lymphoma, resulted in only the low titer of SARS-CoV-2 spike IgG.However, patients had a low titer of SARS-CoV-2 spike IgG response and nasopharyngeal virus clearance, which corresponds to the complete recovery.Based on this, we believe that SARS-CoV-2 VST treatment can still be considered effective.The ratio of CD4 + CD45RO + memory T-cells to VST treatment showed an expansion.In all three cases, CD4 + CD45RO + memory T-cells increased with VST treatment, but the kinetics were occasionally different.The proportion of naive T-cells CD4 + /CD45 RA + decreased with VST treatment and gradually increased between 4 and 8 weeks in all three patients.CD8 + CD45RO + memory T-cells started at different rates at screening in all three patients, and their VST kinetics also differed but stabilized between 30 and 40% in all patients 2 months after VST treatment.Regarding the cytokine levels, the normalization of IL-6 and IL-8 levels for SARS-CoV-2 VST treatment was the best indicator of recovery.
The presence of SARS-CoV-2 RNA, of course, does not prove infectivity.Unfortunately, the viability of the SARS-CoV-2 virus was not determined in the 3 patients.The development of even dangerous mutations during persistence can occur at any time in an immunocompromised patient.There is no correlation whatsoever to determine how viability is related to the emergence of further viral mutations.Therefore, whether it is a viable or a persistent SARS-CoV-2 Vol.: (0123456789) PCR positivity with other mechanisms, it is a clear to achieve PCR negativity, which in our experience is occurred by administration of virus-specific T-cell therapy (3, 4, and 9 weeks after VST administration).
After VST treatment, we were unable to detect VST donor-derived microchimerism in white blood cells or in sorted CD3 + T-cells.This could be due to fact that the amount of 10 3 /kg-10 4 /kg IFNγ + cells was below the detection limit of our ddPCR method (0.05%).This concept was supported by the fact that microchimerism was detected by a ddPCR with sensitivity of 0.01% in phase 1 RELEASE study using CD45RA-depleted memory T-cells in doses 1 × 10 5 -1 × 10 6 /kg produced by CliniMACS Plus® [33].
In our study, we examined not only convalescent donors but also those who had been vaccinated.It was found that an adequate cell content could also be produced from a donor who had received a vaccine.The composition of CliniMACS® Prodigy SARS-CoV-2 VST final product was closely related to the distribution of specific T-cells measured during the screening.At present, the optimal VST dose is heavily debated.In our protocol, allogeneic transplant recipients were given a dose of 5 × 10 3 VST/kg twice over 1-2 week period, while the autologous-HSCT patient with T-cell lymphoma received an increased dose of 1 × 10 4 /kg, considering the immunological milieu of the underlying disease.The U.S. pediatric HSCT group used CliniMACS® Prodigy to treat viral reactivations [40].With HLA-mismatched family donors, 5 × 10 3 CD3 + T-cells/kg and with HLAidentical sibling donors 2.5 × 10 4 CD3 + T-cells/kg VST were applied.We assume that the efficient dose of VST also depends on the production method such as ex vivo expansion, HLA-tetramer, or IFN-γ capture technique.The dose of SARS-CoV-2 VST therapy should also be guided by the proportion of non-IFNγ producing cells, which should be kept below the GVHD threshold, namely, 2 × 10 4 non-IFNγ producing cells/kg.
With CliniMACS® Prodigy products, we do not currently know whether the CD4 + IFNγ + or CD8 + IFNγ + subpopulations were of major clinical importance.In case of cellular products manufactured by a different method, such as AlloVir® T-cell treatment (Viralym-M), it was clearly demonstrated that CD4 + T-cell subpopulation was of importance for the treatment of BK virus hemorrhagic cystitis [41].The outcome of the SARS-CoV-2-specific peptide pool during donor screening could forecast which T-cell subpopulation will dominate the final positive target fraction of the SARS-CoV-2 VST product.In addition to the adoptive T-cell transfer, other therapeutic options for influencing the antiviral T-cell response are being assessed currently, such as recombinant IL-7, low-dose recombinant IL-2, Th1 activators, Th17 blockers, and immune checkpoint inhibitors [20,21,42].
Presently, several cellular therapy studies are underway that use allogeneic CD4 + and/or CD8 + T-cells to treat COVID-19 infection [37].Notably, one of these trials is employing SARS-CoV-2 VSTs, produced via the IFNγ CCS with the CliniMACS® Prodigy system, mirroring the methodology used in our current report (Fig. 4A).In phase 1-2 trial, a dose of 5 × 10   approach, memory T-cells specific to pathogens encountered during the donor's lifetime can be transferred to recipients, which can be of great importance to overcome secondary infections during COVID-19 (Fig. 4B) [31].In CD45-depleted DLI study, the donor eligibility is the same as those for SARS-CoV-2 VST.After apheresis of the convalescent donors, CD45RA + cells undergo immunomagnetic depletion using CliniMACS® CD45RA reagent in CliniMACS® Plus system.A phase 1-2 study by a Spanish working group is ongoing (NCT04578210) investigating memory T-cell DLI from convalescent COVID-19 donors [33].The main selection criteria are COVID-19 pneumonia and/or lymphopenia (< 1.2G/l) and O 2 saturation ≤ 94% oxygen without need for support or ≤ 2.5 l/min through a nasal cannula.In the phase 1 RELEASE study, 9 patients were treated with 3 doses in a distribution of 3-3-3, such as 1 × 10 5 CD45RA-T-cell/kg, 5 × 10 5 CD45RA-Tcell/kg, and 1 × 10 6 CD45RA-T-cell/kg [33,37].At 28 days, all patients recovered.The phase 2 study was conducted at a dose of 1 × 10 6 CD45RA-T-cell/ kg [33].The above experiences provide an opportunity to combat COVID-19 infection by applying allogeneic CD45RA negative memory T-cells, which can contain the very low alloreactive T-cell content (CD45RA + T-cell content 10 2 /kg).
The SARS-CoV-2-specific T-cell infusions produced by ex vivo expansion techniques contain central memory T-cell phenotype T-cells lacking alloreactivity (Fig. 4C).The ex vivo expansion technique provides 100-200 times more cells than products manufactured by other methods, giving the opportunity to build cryopreserved biobanks [34,[45][46][47][48].The ALVR109 off-the-shelf SARS-CoV-2 VST has been administered to 11 COVID-19-infected patients, including 5 as part of clinical trial NCT04401410 and 6 cases as part of the emergency investigational new drug FDA approval [49][50][51].The first successful treatment with SARS-CoV-2 VST from ex vivo expansion technique was described in a moderately severe SARS-CoV-2 delta variant infection in an immunocompromised heart SOT patient [49].Combined remdesivir, tocilizumab, and immunosuppressive therapies were ineffective, and persistent nasopharyngeal swab SARS-CoV-2 PCR positivity was detected [49].The patient received off-the-shelf ALVR109 T-cell infusion from 4/8 HLA-matching donor.Three doses of ALVR109 were used, and nasopharyngeal swab virus clearance was observed already after the first dose.In addition, Vasileiou et al. administered cryopreserved ALVR109 to 4 patients, of which 3 had hematological malignancies (1 Hodgkin's lymphoma, 1 non-Hodgkin lymphoma after autologous-HSCT, 1 chronic myeloid leukemia after allogeneic-HSCT) and 1 elderly patient with hypertension [50].Prior to VST treatment, they were treated with steroid, convalescent FFP, and remdesivir.As a result of VST treatment, 3 patients recovered, and 1 died.Expansion of SARS-CoV-2 reactive T cells was observed in the patients.Haidar et al. gave SARS-CoV-2 VST from convalescent donors to 6 immunocompromised patients (4 lymphomas, 2 after lung transplantation).All patients showed clinical signs and viremia [51].Viral RNA copy number was decreased.On average, 2 doses of 2 × 10 7 cells were used.For VST treatment, 2 patients achieved a complete response, 1 had a sustained response, and 3 patients experienced a partial response followed by death.Overall, 7 out of 11 patients who received ALVR109 VST recovered, resulting in a long-term survival rate of 63% [50,51].Disappointingly, BATIT phase 2 study testing ALVR109 in COVID-19 infection (NCT04401410) had to be terminated early due to difficulties in selection [45].In Viralym-M (Allo-Vir®), phase 2 study provided evidence that it could effectively treat BK virus hemorrhagic cystitis [41].In addition, in a phase II clinical trial, a 92% response rate with AlloVir was demonstrated in immunocompromised patients in case of EBV, CMV, AdV, BKV, and HHV-6 infection or reactivation [34].
In immunocompromised patients with persistent COVID-19, the emergence of multimutational SARS-CoV-2 variants is an important concern [15,17,60].In a young patient with B-ALL, 12 acquired mutations were identified during 3 months of persistent SARS-CoV-2 PCR positivity [61].Furthermore, remdesivir and monoclonal antibody-resistant mutations have been discovered in immunocompromised individuals [62,63].New mutations can result in the emergence of more virulent variants, as well as those with a more pronounced immune escape potential.Therefore, the use of adoptive T-cell therapy in immunocompromised individuals to reach rapid viral clearance is pivotal [64,65].
COVID-19 has acutely highlighted the vulnerability of the elderly due to immunosenescence [66][67][68][69][70].The virus has disproportionately affected older individuals, who are more likely to experience severe symptoms, hospitalization, and death.Immunosenescence is a gradual functional decline of the immune system that is associated with aging, characterized by reduced immune cell function and adaptability, leading to an increased susceptibility to infections, including SARS-CoV-2 [71].In the elderly, the number and activity of T cells and B cells decrease, and their ability to respond to new antigens diminishes.Additionally, the functionality of innate immune cells is also compromised.This deterioration in immune function leads to increased morbidity and mortality from SARS-CoV-2 infection [66][67][68][69][70].The aged immune system's diminished capacity to mount a strong response results in less effective control and clearance of the viral infection, which can in turn lead to more extensive organ damage.The evolutionarily conserved molecular and cellular mechanisms of aging, which contribute to immunosenescence, include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication [71].Genomic instability and telomere attrition contribute to the decreased proliferation capacity of immune cells and the accumulation of dysfunctional cells; epigenetic alterations affect gene expression in immune cells, whereas loss of proteostasis can impair the function of immune proteins, leading to a suboptimal immune responses.Deregulated nutrient sensing and mitochondrial dysfunction affect the energy supply to immune cells, compromising their function.Stem cell exhaustion reduces the body's capacity to replenish the immune system, leading to a decline in immune function over time.Finally, altered intercellular communication can disrupt the coordinated immune response to pathogens.Cellular senescence is a DNA damage-induced cellular stress response characterized by irreversible cell cycle arrest and altered cell morphology and function.Increased cellular senescence with age can lead to an accumulation of dysfunctional immune cells that not only are ineffective in mounting a response to pathogens but can also promote inflammation.Senescent cells exhibit a highly inflammatory senescence-associated secretory phenotype (SASP) characterized by the increased secretion of inflammatory mediators and factors that degrade the extracellular matrix [69].Moreover, senescent cells can also induce senescence in neighboring cells, exacerbating inflammation.Importantly, COVID-19 associates with increased presence of senescent cells (i.e., virusinduced senescence (VIS)), which, combined with the effects of aging propel the development of hyperinflammation and, ultimately, a cytokine storm [71][72][73].Collectively, these mechanisms of aging orchestrate the development of immunosenescence, reducing the effectiveness of the immune system and contributing to the increased morbidity and mortality of COVID-19 in the elderly.With respect to older HSCT recipients, numerous centers have progressively expanded the upper age limit for transplantation, now often accepting patients up to 70-75 years of age.A comparable age expansion is also observable in the case of SOT recipients.Virus-specific T-cell therapy may potentially demonstrate efficacy in these older immunocompromised patients as well, providing a promising treatment avenue for COVID-19 in this vulnerable patient population.Our understanding of the adaptive transfer of allogeneic memory T-cells in immunocompromised/HSCT/SOT recipients highlights the potential for broader clinical applications.Specifically, this method could be utilized effectively in other sub-groups of frail older adults and/or older patients with co-morbidities.
The main limitation of our study is the low number of VST-treated patients.This is primarily due to the laborintensive, time-consuming, and expensive processes involved in establishing this specific cellular therapy.Nevertheless, we have observed that SARS-CoV-2 VST, produced via an interferon-γ cytokine capture system using the CliniMACS® Prodigy device, demonstrates promising clinical efficacy in clearing the SARS-CoV-2 virus.Both convalescent and vaccinated donors can serve as viable sources for SARS-CoV-2 VST, underscoring its versatile application.Through this therapy, we have evidenced not just clinical recovery, but also clearance of the virus itself, suggesting that adoptive T-cell transfer could present a solution for persistent SARS-CoV-2 positivity in immunocompromised hosts.As we look towards the future, the availability of SARS-CoV-2 VST therapy could become a vital instrument in decreasing the number of virus reservoirs and thereby reducing the potential for the emergence of potentially dangerous mutations.However, the expansion of SARS-CoV-2 VST therapy hinges on the outcomes of larger, more extensive clinical trials.Should these trials prove successful, and with the appropriate upscaling of capacity, this treatment could become a viable option for wider use.Of particular note is our conclusion that "off-the-shelf" SARS-CoV-2 VST could serve as a significant resource for elderly, co-morbid, or otherwise immunocompromised individuals.By overcoming immune system dysfunctions and mitigating the consequences of immunosenescence, this therapy may potentially prevent complications, promote healing, and achieve virus clearance.
7-48.29%).In the CD4 + T-cell fraction, the proportion of IFNγ + cells was similar (0.053-0.114%).As opposed to CD4 + T-cell fraction results, the ratio of CD8 + IFNγ + cells showed wider variability (0.064-1.831%).In the target fraction of the Prodigy COVID-19 VST end product, the ratio of CD3 + T cells ranged from 67.713 to 76.345.However, changes in the ratio of T-cell subpopulations, including those showing IFNγ expression, were associated with results of SARS-CoV-2-specific T-cells measured during donor screening.The VST target fraction of the convalescent donor in case 1 contained almost identical proportions of CD4 + IFNγ + (83.71%) and CD8 + IFNγ + (84.61%)T-cells.Similar results were showed in the target fraction of COVID-19 VST product of the vaccine donor in case 2. In accordance with SARS-CoV-2-specific T-cells that are one order of magnitude smaller but balanced compared to the convalescent donor during screening, the VST target fraction contained similar proportions of CD4 + IFNγ + (79.65%) and CD8 + IFNγ + (76.39%)COVID-19 specific T-cells.The convalescent ± vaccinated donor in case 3 showed a high proportion of CD8 + IFNγ + SARS-CoV-2-specific T-cells at screening.Accordingly, the predominance of CD8 + IFNγ + (95.98%)T-cells in the VST-positive target fraction was clear compared to CD4 + IFNγ + (64%) T-cells.The predominance of CD4 + IFNγ + or CD8 + IFNγ + T-cells in the Prodigy SARS-CoV-2 VST target fraction was predicted by the proportion of SARS-CoV-2-specific CD4 + IFNγ + or CD8 + IFNγ + T-cells measured in peripheral blood at donor screening.

Table 1
Clinical characteristics of SARS-CoV-2 VST therapy recipients VST, virus-specific T-cell; AML, acute myeloid leukemia; Ph + B-ALL, Philadelphia chromosome positive acute lymphoid leukemia; HSCT, hematopoietic stem cell transplantation; CR, complete remission; HLA, human leukocyte antigen; NA, not applicable; Thio, thiotepa; Treo, treosulfan; Flu, fludarabine; TBI, total-body irradiation; Eto, etoposide; TEAM, thiotepa, etoposide, cytarabine, melphalan; PBSC, peripheral blood stem cells; GVHD, graft-versus-host disease; PTCY , post-transplant cyclophosphamide; MMF, mycofenolate-mofetil; FFP, fresh frozen plasma; IPS, interstitial pneumonia syndrome; ARDS, acute respiratory distress syndrome; MOF, multiorgan failure; MDS-MLD, myelodysplastic syndrome with multilineage dysplasia Strategies Against SARS-CoV-Two) clinical trial was initiated by Central Hospital of Southern Pest National Institute of Hematology and Infectious Diseases (Budapest, Hungary) and was approved by the Scientific and Research Ethics Committee of the Hungarian National Medical Scientific Council (ETT-TUKEB IV/3937-1/2020/EKU).The SARS-CoV-2 virus-specific T-cell (VST) treatment program was supported by the decision of Ministry of Human Resources (No. IV/4027/2022/EKF).This program was supported by the "Establishment of an expert system to support personalized medicine for managing the care of infectious and major public health diseases" project (grant no.2020-1.1.6-JÖVŐ-2021-00011,"Investment in the future Fund") and by the "Clinical research of COVID-19 disease diagnostics, course of disease and therapy at the Central Hospital of Southern Pest -National Institute of Hematology and Infectious Diseases and in its affiliated institutions" project (grant no.TKP2021-EGA-08, "Thematic Excellence Program 2021").The COVID-19 VST trial was approved by the Scientific and Research Ethics Committee of the Hungarian National Medical Scientific Council (ETT-TUKEB IV/2743-1/2021/EKU).The publication is supported by the "EFOP-1.8.0-VEKOP-17-2017-00001 project."