Adoptive T-cell therapy for virus-associated diseases

SUMMARY

Viral infections remain a significant and predictable challenge in solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients. Although antiviral drugs are commonly used for prophylaxis or early treatment, their long-term use is limited by toxicity, high costs, and the emergence of drug-resistant viral strains, often leading to treatment failure. Cellular immune therapies, particularly adoptive transfer of virus-specific T cells (VSTs), have emerged as a promising alternative, with proven efficacy in controlling hematological malignancies and severe viral infections. While donor-derived VSTs can effectively suppress viral replication in HSCT and SOT recipients, this approach is not feasible when donors are seronegative or inaccessible. A novel single-platform technology now allows for the rapid generation of multi-virus-specific T cells from healthy donors, broadening the applicability of this strategy. In addition, immune monitoring tools can help identify high-risk patients, enabling earlier and more targeted interventions. Emerging data suggest that adoptive T-cell therapy may be used not only therapeutically but also prophylactically, potentially replacing conventional antivirals and reducing adverse effects in immunocompromised patients. This review provides historical foundations and recent advancements in the use of adoptive T-cell therapies for virus-associated complications in transplant recipients.

INTRODUCTION

The human immune system has evolved multiple layers of protection against viral infections. While first-line protection is provided by barriers (e.g., skin and mucosal surfaces) followed by innate immune mediators, long-lived protection is dependent upon the establishment of virus-specific humoral and cellular immune responses (1). The primary mediators of long-lived adaptive immunity are antibodies that neutralize viruses and prevent entry into cells and T cells that eradicate virally infected cells (2–4). During acute infection, this coordinated response leads to the clearance of the virus and protection upon subsequent challenge. The control of viruses that establish persistent latent infection is also mediated by antibodies and T cells (5, 6). While not necessarily preventing the dissemination of latent viruses to a new host, adaptive immunity limits symptomatic disease by rapidly controlling viral reactivation, thus promoting immune equilibrium between the virus and host. Breakdown of this immune equilibrium as a consequence of immunodeficiency can lead to significant uncontrolled viral replication.

Immunodeficiency is most commonly associated with active immunosuppression following organ transplantation or with inborn errors of immunity (7–9). Viral complications remain a significant cause of morbidity and mortality in both settings. Transplant patients are at an increased risk of infectious complications due to the immunosuppressive therapy required to prevent organ rejection (10, 11). These therapies, which include corticosteroids, calcineurin inhibitors, and monoclonal antibodies, suppress the immune response, making patients more susceptible to opportunistic infections. Viral infections are a common and predictable problem in these patients and can lead to end-organ damage and mortality (Fig. 1). Clinical studies indicate that up to 60%–70% of solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) transplant patients experience viral infections post-transplantation (12–14). These infections can lead to prolonged hospital stays, increased healthcare costs, and complications such as secondary bacterial or fungal infections. In addition, certain viruses, such as cytomegalovirus (CMV) and Epstein–Barr virus (EBV), have been linked to chronic immune activation, which can further compromise graft function and patient quality of life (15). Clinical studies have indicated that approximately 30% of infectious deaths after transplantation are associated with primary viral infection or recrudescence of an existing latent infection (16). Antiviral drugs given either prophylactically or as early therapy for patients with detectable viral loads appear to be an effective strategy for reducing viral infections (17, 18). However, long-term treatment with these drugs is associated with significant toxicity, expense, and the appearance of drug-resistant virus isolates, which ultimately results in treatment failure (19). The economic burden of managing viral infections in transplant patients includes costs related to prolonged antiviral therapy, frequent monitoring, and potential retransplantation in cases of severe graft dysfunction (12–14).

Fig 1.

Common viral causes of disease in immunocompromised patients and their target organs.

VIRUS-SPECIFIC T-CELL (VST) THERAPY

The general principle of VST therapy is to restore immune control of virally infected cells through the administration of VSTs. Over the past three decades, VST therapy has been developed to treat hematopoietic stem cell transplant (HSCT) patients, solid organ transplant (SOT) patients, and patients with virus-associated cancers (Fig. 2). It has been used to target a range of viruses, including the common herpesviruses CMV and EBV, the BK and JC polyomaviruses (PyV), adenovirus (AdV), coronavirus (SARS-CoV-2), human papillomavirus (HPV), and hepatitis B virus (HBV). VST therapies have also been developed to target multiple viruses in a single (multi-virus-specific) therapy. VSTs recognize and kill infected cells through the interaction of their unique T-cell receptor (TCR) with viral peptides presented by human leukocyte antigens (HLA) on the surface of infected cells (20, 21). A TCR will typically only recognize with high affinity a single viral peptide epitope presented by a single HLA allele or a family of closely related HLA allele subtypes (22–24). HLAs are classified into HLA class I alleles that typically present intracellular antigens to CD8⁺ T cells, and HLA class II alleles that typically present exogenously captured antigens to CD4⁺ T cells. HLA genes show incredible genetic diversity with currently over 40,000 different human HLA allele sequences; however, much of this is encoded by synonymous changes in the DNA sequence (25). HLA alleles bind different peptide epitope sequences characterized by shared amino acid residues that anchor the peptide to the HLA molecule (26). HLA alleles can be further grouped into supertype families based upon their peptide-binding motif (27, 28). Effective VST therapy needs to be able to accommodate this HLA genetic diversity.

Fig 2.

Proportion of patients who have received VSTs for different viral diseases, by population: HSCT recipients (top panel), SOT recipients (middle panel), and cancer patients (bottom panel). Data were collected from published studies on VSTs.

A number of strategies have been developed for VST therapy (Fig. 3). Early studies used adoptive immunotherapy based on the infusion of unmanipulated donor lymphocytes. This strategy has demonstrated some clinical efficacy in the treatment of multiple viral complications, including EBV-associated post-transplant lymphoproliferative disorder (PTLD), CMV, AdV, and human herpesvirus 6 (29–32). However, adoptive immunotherapy with unmanipulated donor lymphocytes is associated with a high risk of graft-versus-host disease (GvHD) due to a high frequency of alloreactive T cells (33, 34). Subsequent studies have shown that depletion of naïve T cells (through depletion of CD45RA⁺ cells) can reduce the risk of GvHD in patients treated with unmanipulated donor lymphocytes (31, 35). One of the major limitations of this approach is that unmanipulated donor lymphocytes often contain a low frequency of VSTs, which can lead to inconsistent clinical responses. Two broad strategies have been developed to overcome these limitations. These include either ex vivo enrichment of VSTs or ex vivo expansion of VSTs following stimulation with viral antigens or peptide epitopes (Fig. 4). These strategies allow selective enrichment of VSTs that target the antigen in question.

Fig 3.

Adoptive immunotherapy approaches used to treat viral infection in immunocompromised patients.

Fig 4.

Ex vivo expansion vs ex vivo enrichment for the generation of VSTs.

Ex vivo enrichment of VSTs usually involves two distinct methods—HLA-peptide multimer selection and cytokine (interferon gamma; IFNγ) capture. HLA-peptide multimer enrichment has been successfully used to either treat or prevent multiple viral infections, including CMV, AdV, and EBV (36, 37). More recently, HLA-peptide Streptamer enrichment technology has allowed more efficient selection of VSTs (38–41). Streptamer technology allows dissociation of the HLA-peptide complexes from T cells prior to adoptive immunotherapy, allowing these T cells to retain their functional and phenotypic profile. Adoptive transfer of Streptamer-enriched T cells from HSCT donors or third-party donors has a good safety profile with minimal acute/chronic GvHD (38–41). One limitation of HLA-peptide multimer technology is its application to only defined T-cell epitopes and a restricted number of HLA alleles. An alternative strategy that overcomes this limitation is to enrich VSTs using IFNγ-capture technology. In this approach, T cells in peripheral blood mononuclear cells (PBMC) are stimulated with synthetic peptides or viral antigens, and then the cells expressing IFNγ following antigenic stimulation are enriched using magnetic beads. A number of clinical studies have shown that IFNγ capture technology can be consistently used for enriching T cells specific for individual viruses (CMV and EBV) and multiple viruses (CMV, EBV, and AdV) with minimal side effects (42–53). Most of the patients treated with these T cells showed complete or partial responses, including resolution of viral reactivation and end-organ disease. Here again, the ex vivo enrichment has limited application to those viral infections where the ex vivo T-cell frequency is high. Indeed, antigen-specific T-cell precursors specific for BKPyV and JCPyV are too low to allow reproducible enrichment.

In vitro expansion of VSTs using synthetic peptide epitopes, viral vectors, or virus-infected cells offers a more efficient strategy to provide adoptive T-cell therapies for viral complications. These in vitro expansion strategies include highly focused approaches that generate T-cell clones that recognize a single HLA-peptide complex (54–56) and broader approaches that use antigen-expressing professional antigen-presenting cells to stimulate polyclonal VST populations (57–60). Most current approaches under development use pools of synthetic peptides that encode either overlapping peptides from immunodominant antigens or well-characterized HLA-restricted peptide epitopes (61–64). While population coverage may be impacted using the latter approach due to the worldwide diversity of HLA, it provides a more amenable approach to VST manufacturing and quality control. In addition, a number of common HLA alleles are prevalent in most worldwide populations (Table 1), providing an opportunity to target T cells recognizing peptides presented by these common alleles. The effective application of VST therapy requires a donor source from which to generate VSTs. While some strategies have been developed to generate VSTs from material obtained from unexposed individuals, such as umbilical cord blood (65), the most effective approaches rely upon the use of peripheral blood from volunteers who have developed virus-specific T-cell memory. In HSCT recipients with viral disease, seropositive donors provide an effective cell source for VST expansion (54, 58). However, this approach is not applicable for patients who receive an HSCT from a seronegative donor or for SOT recipients. In both settings, autologous VST therapy has been reported in case studies and early phase trials (59, 60, 66, 67). Autologous VST therapy has also been studied extensively for the treatment of virus-associated malignancies (68–76). Despite these advances, autologous VST therapy faces significant hurdles. Autologous VSTs cannot be generated from all patients and may have reduced potency due to the underlying immunosuppression in the patient. The generation and quality testing of autologous VSTs takes multiple weeks, which can limit their use, as patients frequently succumb to progressive disease before the T cells are ready for infusion. Current approaches have therefore moved to a VST therapy that can be offered as an allogeneic “off-the-shelf” treatment (63, 77–81). Allogeneic VST therapy relies upon the generation of a repository of T cell products from a selected cohort of individuals (Fig. 5). Allogeneic VSTs offer a number of advantages over autologous VSTs (Fig. 6). These VST donors are selected to provide broad HLA coverage within a given population, and therapeutic products are generated to offer multiple doses to multiple patients (82). These VSTs can also be administered within days of patient recruitment and selected based upon algorithms that match the global HLAs between the VST product and patient, and the HLA-restricted virus specificity of the product (82). Current approaches now incorporate multiple virus specificity in VSTs, which allows the targeting of multiple viral complications in a patient using a single T-cell product (62, 63, 65, 77, 81, 83–96). Multicenter clinical trials of VSTs in adult and pediatric patients have demonstrated potential clinical benefit ( Table 2). It is important to highlight that clinical studies have shown that allogeneic VSTs are well tolerated with minimal side effects, including acute toxicities, which are generally associated with the administration of donor lymphocyte infusions. Following allogeneic or autologous VST infusions, there has been a negligible incidence of grade 2–4 acute or chronic GvHD, and no instances of graft or organ rejection have been recorded. In addition, cytokine release syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS), which are often observed following CAR T cell therapies (97), have rarely been documented following adoptive immunotherapy with VSTs directed to either a single virus or multiple viruses (98). Interestingly, recent clinical studies with allogeneic EBV-specific T cells expressing CD19 CAR showed complete responses in 70% of patients with relapsed/refractory B-cell malignancies and no evidence of CRS or ICANS (99). These observations further emphasize the safety of allogeneic VSTs in clinical settings.

TABLE 1.

Common HLA alleles prevalent in most worldwide populations

Population	HLA class I alleles	HLA class II alleles
Caucasian	A*02:01, A*01:01, A*03:01, A*24:02, B*07:02, B*08:01, B*44:02, B*35:01, C*07:01, C*04:01	DRB1*15:01, DRB1*03:01, DRB1*04:01, DRB1*07:01, DQB1*06:02, DQB1*02:01, DQB1*03:01, DQA1*01:02, DQA1*05:01, DPB1*04:01
Black (African)	A*30:01, A*02:01, A*23:01, A*68:02, B*53:01, B*58:01, B*15:03, B*42:01, C*06:02, C*17:01	DRB1*13:02, DRB1*11:01, DRB1*03:01, DRB1*15:03, DQB1*06:02, DQB1*05:01, DQB1*02:01, DQA1*05:01, DQA1*01:02, DPB1*01:01
Asian (East Asian)	A*24:02, A*11:01, A*02:01, A*33:03, B*15:01, B*40:01, B*46:01, B*58:01, C*01:02, C*07:02	DRB1*09:01, DRB1*12:02, DRB1*15:01, DRB1*04:05, DQB1*03:03, DQB1*06:01, DQB1*03:01, DQA1*01:03, DQA1*03:01, DPB1*05:01
Hispanic	A*02:01, A*24:02, A*03:01, A*68:01, B*35:01, B*44:03, B*07:02, B*15:01, C*07:02, C*04:01	DRB1*04:07, DRB1*07:01, DRB1*08:02, DRB1*03:01, DQB1*03:02, DQB1*02:01, DQB1*03:01, DQA1*05:01, DQA1*03:01, DPB1*04:02
South Asian	A*02:01, A*11:01, A*33:01, A*24:02, B*35:01, B*40:06, B*15:01, B*51:01, C*07:01, C*04:01	DRB1*15:01, DRB1*07:01, DRB1*03:01, DRB1*13:01, DQB1*06:01, DQB1*02:01, DQB1*03:01, DQA1*01:02, DQA1*05:01, DPB1*02:01

Fig 5.

Schematic of autologous vs allogeneic manufacture of VSTs.

Fig 6.

Benefits of allogeneic VST products.

TABLE 2.

Multicenter clinical trials of VST therapies^a

Trial name/product	Phase/design	Target viruses/disease	Population	References
Third-Party VST Bank Trial	Phase 1/2, multicenter	EBV, CMV, AdV	Allogeneic HSCT recipients with refractory infections	(77)
Posoleucel (ALVR105)	Phase 2, multicenter, open-label	AdV, BKPyV, CMV, EBV, HHV-6, JCPyV	Allo-HCT recipients	(61, 95, 100)
ALVR106	Phase 1, multicenter, double-blind	Respiratory viruses (e.g., RSV, influenza, PIV, hMPV)	HCT and SOT recipients with respiratory viral infections	(101)
ALLELE Trial (Tabelecleucel)	Phase 3, multicenter, open-label	EBV (PTLD)	SOT or HSCT recipients with relapsed/refractory EBV +PTLD	(81)
AdV-specific T cells (Pediatrics)	Phase 1/2, multicenter	AdV	Pediatric post-HSCT patients	(102)
AdV-VSTs from Donors	Phase 1/2, multicenter	AdV	Post-HSCT patients	(103)
ACES	Phase 2, open-label	CMV, EBV, or BKPyV	Post-HSCT patients	(62)
IVIST01 Trial	Phase 1/2, randomized, multicenter, open-label	CMV, AdV, HSV (immune monitoring guided therapy)	Pediatric kidney transplant recipients	(104)
MULTIVIR-1	Phase 1/2, multicenter, randomized	CMV and EBV	Post-HSCT patients	(105)
VANCE	Phase 3, randomized, multicenter	EBV (nasopharyngeal carcinoma)	Nasopharyngeal carcinoma patients	(106)
EMBOLD	Phase 1/2, multicenter, open-label (Phase 1), randomized (Phase 2)	EBV (progressive multiple sclerosis)	Progressive multiple sclerosis patients	(107)

^a AdV: adenovirus, BKPyV: BK polyomavirus, CMV: cytomegalovirus, EBV: Epstein–Barr virus, HHV-6: human herpesvirus 6, hMPV: human metapneumovirus, HSCT: hematopoietic stem cell transplant, HSV: herpes simplex virus, JCPyV: JC polyomavirus, PiV: parainfluenza virus, PTLD: post-transplant lymphoproliferative disease, SOT: solid organ transplant, VST: virus-specific T cell.

LATENT VIRUSES

EBV

EBV is a lymphocryptovirus with tissue tropism for human B cells (108, 109). Infection occurs in early childhood or during adolescence (110–112). Exposure typically occurs via bodily fluids or, more rarely via direct transmission of EBV-infected cells following organ transplantation. Following transmission by saliva via the oropharyngeal epithelium, EBV infects B cells in Waldeyer’s tonsillar ring (113, 114). It then induces the coordinated differentiation of B cells that promotes transitioning from a highly proliferative state through a germinal center-like state to a quiescent memory-like state (115). The EBV genome is maintained in episomal form and tethered to the host genome, allowing the spread of the viral genome into daughter cells upon homeostatic cellular division (116). Lytic EBV reactivation is associated with B-cell activation and maturation into a plasma cell-like state (117). EBV encodes around 100 open reading frames, the majority of which are associated with the lytic phase of infection (118). Latency is regulated by a small subset of genes, the EBV nuclear antigens (EBNA) 1-6, and the latent membrane proteins (LMP) 1 and 2. Latency gene expression is tightly regulated with the phases of EBV latent infection (118). The proliferative phase of expression is associated with a latency III profile and the expression of EBNA1–6 and LMP1&2. The latency II germinal center phase is associated with expression of EBNA1 and LMP1&2, while the quiescent memory-like state is associated with latency I/0 and the expression of EBNA1 only (119).

EBV-associated diseases

Diseases associated with EBV infection can be defined by the stage of infection, the immunocompetency of the infected individual, and a well-defined or undefined role of EBV in disease manifestation. In immunocompetent individuals, primary acute lytic infection is usually asymptomatic and self-limiting (120). However, in adolescents and young adults, it is the primary cause of infectious mononucleosis (120–123). Primary disease can also be associated with more severe complications, particularly in the setting of primary immunodeficiency, including X-linked lymphoproliferative disease and chronic active EBV disease (124–126). In the transplant setting, immunosuppression can lead to the uncontrolled proliferation of EBV-infected B cells, resulting in PTLD (125, 127–129). In immunocompetent individuals, EBV is present in a range of malignancies, including but not limited to Hodgkin lymphoma, diffuse large B-cell lymphoma, NK/T cell lymphoma, nasopharyngeal carcinoma, and gastric carcinoma (130–133). Between 150,000 and 350,000 new cases of EBV-associated malignancies occur each year (134, 135). These diseases have a defined role for EBV in their manifestation, and the potential for EBV-based immune interventions is well established. However, the role in the manifestation of other diseases is not well defined. EBV is now believed to have a causative role in multiple sclerosis (MS) and has been associated with other autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, and Sjögren’s syndrome (136–139).

VSTs and EBV-positive PTLD

EBV-positive PTLD occurs in HSCT and SOT patients. PTLD is predominantly associated with the EBV latency III proliferative phenotype whereby cell proliferation and survival are maintained by the expression of EBNA1–6 and LMP1&2 (140, 141). A lack of effective immunosurveillance of EBV-infected B cells by EBV-specific T cells prevents the establishment of the normal immune equilibrium that occurs in immunocompetent individuals (142–144). In the HSCT setting, EBV-seropositive recipients who receive an allogeneic transplant from an EBV-seronegative donor or who receive a T-cell-depleted allogeneic transplant have an elevated risk of developing EBV-positive PTLD (145, 146). In the SOT setting, EBV-seronegative recipients have a heightened risk of developing PTLD following transplant from an EBV-seropositive donor (147). Risk also varies in different SOT settings and can also be attributed to differences in HLA matching between recipient and donor (148). EBV-associated lymphoproliferative diseases may also arise in other settings of immunosuppression, including patients with autoimmune diseases (149).

Diagnosis of EBV-positive PTLD has not typically relied upon the monitoring of EBV DNA load, nor is immune monitoring currently approved for the assessment of EBV immunity in patients at risk of PTLD (150). However, more recent studies have shown that EBV-DNAemia >10,000 IU/mL can be used as a strong predictor for EBV-PTLD (151). As there are currently no approved EBV-targeted antiviral therapies, the current standard-of-care treatment for PTLD is the reduction of immunosuppression followed by rituximab. Subsequent lines of therapy then rely upon radiotherapy and chemotherapy. The rate of refractory EBV-positive PTLD varies between transplant types and patient cohorts. It is in the setting of pediatric and adult refractory EBV-positive PTLD that the European Medicines Agency recently approved the use of Ebvallo (tabelecleucel) (81).

The treatment of EBV-positive PTLD has provided the most compelling evidence for the efficacy of VST therapy. Two key findings paved the way for the development of EBV-specific T-cell therapy. First, the demonstration that EBV can stably transform B cells in vitro (152), leading to the generation of EBV-positive lymphoblastoid cell lines (LCL). Second, the demonstration that the outgrowth of EBV-positive LCL was controlled by T cells (153, 154). This led to the development of a T-cell manufacturing process that utilizes EBV-positive LCL to stimulate autologous EBV-specific T cells (155). The initial approaches relied upon repeated stimulation with irradiated EBV-positive LCL over a period of 4–6 weeks. This process typically generates a polyclonal population of predominantly CD8⁺ T cells dominated by EBNA3–6-specific cells. This approach was initially developed for prophylactic and therapeutic use in HSCT patients using T cells derived from EBV-seropositive donors (57, 58, 156–158). Over 450 HSCT patients have now been treated with EBV-specific T cells (Fig. 7). The therapeutic administration of EBV-specific T cells has consistently shown evidence of clinical responses in HSCT recipients, while EBV reactivation is rare following the prophylactic administration of VSTs (Fig. 8).

Fig 7.

Number of HSCT or blood cancer (BC) patients treated with VSTs. The top panel represents the number of patients treated with either ex vivo-enriched or ex vivo-expanded VSTs. The middle panel represents the number of patients treated with an autologous or an allogeneic T-cell product. The bottom panel represents the number of patients treated therapeutically or prophylactically with VSTs. Data were collected from published studies on VSTs.

Fig 8.

Response to therapy in HSCT or blood cancer (BC) patients treated with VSTs. The top panel represents the proportion of patients who had a clinical response (either a complete or partial response) following treatment with VSTs. The bottom panel represents the proportion of patients who had viral reactivation following prophylactic administration of VSTs. Data were collected from published studies on VSTs.

While donor-derived EBV-specific T cells have provided strong evidence of efficacy in HSCT patients with PTLD, their use is limited to HSCT recipients who receive a transplant from EBV-seropositive donors. To overcome this limitation, autologous EBV-specific T-cell therapies were initially assessed in SOT recipients with some evidence of efficacy (59, 60, 159). However, these are often difficult to generate from heavily immunosuppressed patients. A more practical solution has been the development of repositories of EBV-specific T cells generated from cohorts of healthy EBV-seropositive volunteers (29, 160–167). These T-cell repositories have been generated in multiple locations around the world and contain up to 300 EBV-specific T-cell products (50, 78, 81, 168). Similar to HSCT, banked allogeneic EBV-specific T cells are HLA-matched to PTLD patients, although with less stringent matching requirements. Typically, a minimum of two HLA alleles is required for matching, with at least one showing EBV-specific T-cell reactivity. Initial studies suggested that the level of HLA matching might impact clinical outcome (161); however, more recent observations have suggested that this is not necessarily the case (78). The majority of SOT patients who have received VSTs for EBV-associated diseases have been treated with allogeneic VSTs (Fig. 9). Clinical response rates in SOT patients are comparable to those observed in HSCT patients (Fig. 9).

Fig 9.

SOT patients treated with VSTs. The top panel represents the number of patients treated with an autologous or an allogeneic T-cell product. The bottom panel represents the proportion of patients who had a clinical response (either a complete or partial response) following treatment with VSTs.

VSTs and other EBV-positive cancers

EBV-specific T-cell therapy has been assessed against EBV-associated epithelial cancer and lymphoma (Fig. 10). In these immunocompetent settings, EBV-specific T-cell therapy has not yet proven to be efficacious. Escape from immunosurveillance by EBV-positive cancers in these settings is associated with a restricted pattern of EBV latent antigen expression (169–171) and an immunosuppressive tumor microenvironment (172–175). Antigen expression is limited to LMP1&2 and EBNA1 in malignancies with a latency II pattern, and only EBNA in latency I malignancies. There has been some evidence for either complete or partial tumor responses in patients with latency II EBV-associated malignancies (70, 71, 176–182). These objective clinical responses appear more evident in patients with lymphoma. However, a recent phase 3 study in nasopharyngeal carcinoma demonstrated no benefit of EBV-specific T-cell immunotherapy in combination with standard chemotherapy (106). One limitation of the EBV-positive LCL approach for T-cell manufacture to treat latency II malignancies is the preferential expansion of T cells that target EBNA3–6, which are not expressed in latency II malignancies. Other strategies have been developed to preferentially expand T cells specific for latency II antigens (69, 72, 74, 183, 184). While these T cells have been tested in early-phase clinical trials, pivotal trials have not been undertaken to assess their efficacy.

Fig 10.

Virus-associated cancer patients treated with VSTs. The top panel represents the number of patients treated with an autologous or an allogeneic T-cell product. The middle panel represents the proportion of patients with a clinical response (either a complete or partial response) or stable disease following treatment with VSTs. The bottom panel represents the proportion of patients who had disease relapse following administration of VSTs, either prophylactically or as an adjunct to standard of care treatment. Data were collected from published studies on VSTs.

EBV-VST therapy and autoimmunity

A less well-defined role for EBV in the causality of autoimmune disorders has meant that EBV-specific therapeutics have not been extensively studied. A number of clinical trials have commenced investigating antiviral therapies in MS patients (185, 186). One longstanding hypothesis for the role of EBV in MS is a consequence of the accumulation of EBV-infected B cells in the brains of MS patients. We therefore initiated an open-label phase I clinical trial using autologous EBV-VSTs in patients with progressive MS (187, 188). The therapy was well tolerated, and there was evidence in some patients of an association between the quality of the T-cell product and clinical improvement. While follow-up trials using autologous VSTs in MS patients have not yet commenced, studies have been undertaken using allogeneic EBV-VSTs in progressive MS patients. Although the phase 1 results from this approach were promising, no efficacy was observed in a phase 2 clinical trial, and subsequent plans for a phase 3 pivotal study were abandoned (189). Progressive MS is a debilitative stage of disease with few treatment options, and it is probable that an effective EBV-specific therapy will need to be employed at an earlier disease state. A more comprehensive understanding of the role of EBV and EBV-specific immune dysfunction in MS and other autoimmune disorders is also likely required to effectively employ EBV-specific therapies (190).

CMV

CMV is another member of the human herpesvirus family that persists for life following primary infection (191). Similar to EBV, exposure typically occurs via bodily fluids or via direct transmission following organ transplantation (192). It is also transmitted directly from mother to fetus (193). Primary infection is quite pervasive, with infection occurring in endothelial and epithelial tissues throughout the body. CMV establishes latency in cells of the monocytic lineage (monocytes, macrophages, and dendritic cells); however, unlike EBV, it does not appear to induce cellular transformation (194, 195). Viral reactivation typically occurs as a consequence of inflammation-driven cellular differentiation, whereby maturation of the host cell induces lytic gene expression (196). CMV has been described as a “smoldering” infection, in which frequent viral reactivation is kept under control by the immune system (197). However, maintaining immune equilibrium with CMV appears to require significant immunological resources, which is associated with the long-term maintenance of a large memory response and the inflation of the CMV-specific T-cell and antibody responses over time (198–201).

While CMV encodes over 200 open reading frames, T-cell immunity against CMV is primarily directed toward a small subset of immunodominant antigens associated with the lytic cycle of infection (202–204). Predominant among these are phosphoprotein (pp) 65, a major tegument protein, and immediate early (IE) 1, which plays a key role in reactivation from latency. Immunodominant responses against pp65 and IE-1 are found in the majority of CMV-seropositive individuals. Distinct from EBV, T-cell responses against latent-cycle antigens are subdominant and do not appear to play a significant role during persistent CMV infection (205).

CMV-associated diseases

CMV is one of the most important infectious pathogens in clinical transplantation. Acquisition of primary CMV infection or reactivation of latent infection causes significant morbidity and can cause deleterious effects on engrafted organs (206, 207). CMV disease can manifest in many human organs, including the lungs, kidneys, heart, liver, gastrointestinal tract, eyes, and brain. Extensive studies in healthy virus carriers have indicated that immune control of CMV infection is dependent on innate and adaptive immune responses (208–210). Impairment of these immune regulatory pathways due to prolonged immunosuppression, antibody-mediated depletion of T cells, and induction of alloreactive immunity due to MHC mismatch can disrupt the balance between the host immune system and virus (207). Studies have shown that invasive CMV disease affects more than one-third of seronegative recipients who receive a transplant from a seropositive donor (211). This donor/recipient combination creates the highest risk of CMV disease in the recipient, while seronegative recipients of seronegative donors have the lowest risk, and seropositive recipients have medium risk. Several studies have demonstrated that early ganciclovir therapy (antiviral prophylaxis) reduces the incidence of early CMV pneumonitis from 13% to 2%, and the overall rate of CMV disease from 33% to 6% (212). However, the major health cost of this aggressive approach is due to a syndrome referred to as “late CMV,” in which patients develop disease after the completion of prophylaxis, rather than in the engraftment period (typically in the first 2 months). Late CMV is seen in as many as 5%–18% of all at-risk patients, depending on the cohort studied (213). This effect may be due to an attenuated form of the disease, myelosuppressive toxicity of ganciclovir, cessation of ganciclovir at 100 days, and according to more recent data, the direct immunosuppressive effect of ganciclovir (214). More importantly, drug resistance is an increasing concern for patients undergoing long-term ganciclovir therapy (215).

Treatment of CMV disease using VST therapy

The development of CMV-specific T-cell immunotherapy was driven by the emergence of CMV disease as a serious complication associated with immunosuppression following organ transplantation. Riddell and colleagues were the first to demonstrate that CMV-specific T cells could be adoptively transferred to bone marrow (BM) transplant recipients (54, 55). The initial protocol that was established employed pp65-specific T-cell clones that were isolated and expanded from BM donors. This was followed by work from several groups using polyclonal CMV-specific T cells derived from BM and HSCT donors to treat CMV disease in recipients (42, 216–224). The majority of these approaches have used ex vivo-expanded CMV-specific T cells, although ex vivo enrichment has been used in a number of patients (Fig. 7). CMV-specific T cells are maintained at a high frequency in circulation, and VSTs are therefore straightforward to manufacture from the PBMC of CMV-seropositive HSCT donors. Clinical responses have been evident following use of ex vivo-enriched and ex vivo-expanded VSTs (Fig. 8). Autologous CMV-specific T-cell therapy has been employed less frequently to treat HSCT recipients who received a transplant from a CMV-seronegative donor. However, case reports have shown an association with the resolution of CMV disease (225–227). We demonstrated that an autologous VST approach can be used to treat SOT patients with CMV-associated complications, overcoming perceived risks of organ rejection following VST therapy (67). The therapy was well tolerated and associated with improved CMV-specific T-cell immunity.

Following the success of off-the-shelf allogeneic VSTs specific for EBV, CMV-specific T-cell repositories have been developed. Initially, these targeted CMV alone; however, recent work has moved toward the incorporation of CMV-specific T cells in multi-virus-specific VST products. The most comprehensive report on the use of allogeneic VST therapy for CMV, a phase 1/2 study in HSCT patients from Prockop et al., demonstrated a clinical response rate of 64% using pp65-VSTs (79). A number of other studies have seen similar responses in patients with active CMV (77, 92, 228–231). Prophylactic administration of VST therapy for CMV in HSCT recipients has also been reported to reduce the risk of CMV reactivation (90, 93, 94); however, a recent phase 3 study from AlloVir using posoleucel, a multi-virus-specific T-cell therapy, failed to reach its primary endpoint of preventing clinically significant infection, despite promising observations in its phase 2 study (100). CMV-specific allogeneic T-cell approaches have been used less frequently in SOT patients when compared to VSTs for EBV (Fig. 9). We recently reported on the compassionate use of allogeneic VSTs in SOT recipients, demonstrating evidence of a response in 4 of 5 adult patients and one pediatric patient treated for CMV complications (63), and have an ongoing phase 1 study using allogeneic VSTs to treat SOT patients with CMV and other viral diseases.

CMV and cancer

A number of studies have reported the presence of CMV in a range of human cancers (232–234). These observations remain controversial, with no definitive evidence that CMV can cause cellular transformation or that CMV can be isolated from cancer cells. Nevertheless, evidence of CMV DNA, particularly in glioblastoma (GBM), has led to the initiation of studies using CMV-specific therapies, including antivirals and CMV-VSTs in GBM patients (Fig. 10). GBM patients have very poor overall survival, with a median survival of 14 months following primary diagnosis and 9 months following recurrence. The 5-year survival rate for GBM is 5%. Our group reported the first use of autologous CMV-VSTs in recurrent GBM patients using a VST manufacturing process consistent with the approaches used to treat disease in transplant patients (75). Follow-up analysis from this study reported long-term survival (>33 months) in 4 of 12 patients treated with CMV-VSTs (235). Two of these patients survived for more than 5 years. We have subsequently completed an autologous CMV-VST trial in patients with primary GBM (76). Other groups have also reported the use of CMV-specific T cells in patients with GBM (236, 237). To expedite the delivery of CMV-VSTs to GBM patients, our group has moved to an allogeneic CMV-VST approach, which we are currently investigating in a phase 1 study in combination with the checkpoint inhibitor pembrolizumab.

Polyomaviruses

The human polyomaviruses are a family of DNA viruses that establish lifelong latency. All contain a similar genome structure characterized by a small double-stranded DNA genome that encodes three viral capsid proteins (VP1, 2, and 3) and two viral replication proteins, large T antigen (LTA) and small T antigen (STA) (238). Primary infection is typically asymptomatic or associated with mild symptoms (239). This is followed by lifelong persistent latent infection and intermittent viral shedding. Persistent infection is also typically asymptomatic, with viral burden controlled by antibody responses that target the viral capsid proteins and T-cell responses directed toward the viral capsid and replication proteins (240–243). However, polyomaviruses are associated with a significant disease burden in settings of immunocompromise. Most prevalent among these are the two closely related viruses, BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) (244, 245). There are currently no antiviral therapies approved for polyomavirus-associated diseases (246).

BKPyV-associated diseases and VST therapy

BKPyV transmission occurs via a respiratory or fecal-oral route (247–249). Similar to other persistent viruses, it can also be transmitted following organ transplantation (250). BKPyV primarily establishes latency in the epithelium of the kidney and urinary tract (251–253). BKPyV-associated nephropathy (BKVAN) following kidney transplantation is the most common clinical manifestation of BKPyV infection (244). BKVAN occurs in up to 7% of kidney transplant recipients (254). It is associated with a progressive decline in kidney function and can result in loss of the graft kidney in up to 15% of patients (255). BKPyV is also a cause of hemorrhagic cystitis (HC) in HSCT recipients (244, 256). Deficiencies in humoral and cellular immune responses have been implicated in the development of BKVAN and HC (257, 258). Studies have shown that exposure to a different BKV genotype can lead to escape from viral neutralization in kidney transplant recipients with pre-existing immunity (258). However, as with most latent infections, virus-specific T cells appear to play the most critical role in preventing uncontrolled BKPyV reactivation (256, 259). Deficiencies in BKPyV-specific CD4⁺ and CD8⁺ T-cell function and frequency have been associated with an increased risk of BKV viremia and disease (260). The current strategy of reducing the levels of immunosuppression to treat BKPyV has shown some impact on the quality of T-cell immunity in transplant patients (256, 261); however, this approach may come with the increased risk of graft rejection (262). Therefore, BKPyV-specific VSTs have been developed as an alternative therapeutic approach.

In contrast to CMV and EBV, against which a large pool of circulating memory T cells is maintained, the frequency of circulating BKPyV-specific T cells is low (242). Nevertheless, this has not prevented the development of BKPyV-VSTs using approaches that were initially developed for CMV and EBV (85, 86, 263–265). The primary antigenic targets used for BKPyV-VSTs have been VP1, LTA, and STA (242). Clinical studies have been conducted using BKPyV-VSTs expanded from HSCT donors via stimulation with overlapping peptide pools to treat patients with BKPyV viremia or HC (89). The overall response rate was 86% in patients treated for BK viremia, 100% in patients treated for HC, and 87% in patients treated for both BKPyY viremia and HC (89). Distinct from CMV and EBV, BKPyV-VSTs are predominantly comprised of CD4⁺ T cells; however, specific CD8⁺ T cells are present in BKPyV-VST products (63). With the advent of allogeneic cellular therapy, studies in patients with BKVAN and HC have been undertaken using repositories of allogeneic BKPyV-VST products, including multi-virus platforms (63, 85, 86, 89, 90, 92, 94, 231). HSCT recipients (Fig. 7 and 8 ) and SOT recipients (Fig. 9) have been treated with allogeneic VSTs for BKPyY-associated diseases. While promising in early phase 1/2 trials, recent setbacks in a phase 3 study of patients using the allogeneic T-cell product posoleucel from AlloVir indicate that more research is needed to improve the potency of BKPyV-VSTs. A better understanding of the immunodominant epitopes encoded within BKPyV and the specific targeting of these epitopes for T-cell expansion should help to improve the potency of BKPyV-VSTs by promoting an increased proportion of BKPyV-VSTs in the product and improving HLA matching of VSTs to the recipient.

JCPyV-associated diseases and JCPyV-specific T-cell therapy

JCPyV is closely related to BKPyV, with a 72% overlap in genome sequence (266). JCPyV has a distinct tissue tropism from BKPyY, with infection of glial cells, oligodendrocytes, and astrocytes, and less common infection detected in the kidney (252, 267–270). The primary clinical manifestation of JCPyV infection is progressive multifocal leukoencephalopathy (PML). PML is a demyelinating disease of the central nervous system associated with active JCPyV infection and extensive immune infiltration. Initially identified in association with primary B-cell lymphoproliferative disorders (271, 272), PML became more common during the acquired immunodeficiency syndrome epidemic (273). The incidence of PML in patients with human immunodeficiency virus (HIV) has reduced since the advent of highly active antiretroviral therapy (274). More recently, the introduction of new immunosuppressive therapies in patients with autoimmune disease has resulted in an increased risk of JCPyV-associated PML. This is particularly evident in MS patients treated with natalizumab, a monoclonal antibody specific for the alpha-4-integrin (275–277). By preventing T cells from crossing the blood–brain barrier, natalizumab reduces the risk of MS relapse by restricting access of autoimmune lymphocytes that are involved in the pathogenesis of MS (278–280). However, this also leads to the sequestration of JCPyV-specific T cells in peripheral blood, limiting immune surveillance of JCPyV in the brain (281).

In the absence of approved antiviral drugs, the current treatment option for PML is the reduction of immunosuppression to restore JCPyV-specific T-cell immunity (282). Immune-checkpoint inhibition targeting programmed death receptor 1 on the surface of T cells has shown efficacy in patients with PML, reducing JCPyV loads and improving clinical symptoms (283, 284); however, checkpoint inhibition is likely to be contraindicated in patients with autoimmune disorders. The genetic similarity between JCPyV and BKPyV has provided an opportunity to develop VSTs that target both viruses. A few studies have demonstrated that VSTs generated to target BKPyV can also be used in patients with PML (285, 286). While often reported in case reports for compassionate use (287–290), the most comprehensive study to date using close relatives to generate allogeneic BKPyV-VSTs for patients with PML demonstrated that this approach was well tolerated, with no severe adverse events detected (291). These studies report evidence of potential clinical benefit with reduced mortality in HSCT recipients (Fig. 8) and SOT recipients (Fig. 9), although no later phase studies have been undertaken to assess efficacy. VSTs for JCPyV are now being developed as part of allogeneic multi-virus-specific VST repositories (63, 95). Our recent observations have demonstrated similar potential of these multi-virus-specific VSTs in treating patients with PML (63).

Human papillomaviruses

Most human virus-associated cancers are caused by human papillomaviruses, particularly the HPV genotypes 16 and 18 (292, 293). Prevalent worldwide, HPV is associated with cervical cancer, anal cancers, vaginal cancers, vulvar cancers, penile cancers, and oropharyngeal cancers (134). HPV-associated cancers are associated with a lack of efficient immunosurveillance of persistent infection in basal keratinocytes. While most HPV-associated cancers occur in otherwise immunocompetent individuals, HSCT and SOT patients are at an increased risk of developing HPV-associated cancers (294, 295). HPV has a small dsDNA genome encoding eight genes, including L1 and L2 that encode the virus-capsid proteins, and E1, E2, E4, E5, E6, and E7 that are involved in viral DNA replication. While in cervical cancer, protein expression is predominantly limited to E6 and E7 (296), oropharyngeal cancers can demonstrate a broader expression profile characterized by expression of other E proteins (297, 298). T-cell responses in healthy individuals who have cleared HPV infection are typically low to undetectable in circulation; however, persistent infection is associated with detectable T-cell responses against HPV proteins (299, 300). This includes CD4⁺ and CD8⁺ T cells that are elevated during active disease and decline following disease resolution. Analysis of tumor-infiltrating lymphocytes has demonstrated the presence of T cells specific for multiple antigens infiltrating HPV-positive cancers.

Despite the prevalence of HPV-infected cancers worldwide, HPV-specific VST therapy is less advanced, with fewer cancer patients treated with VSTs compared to the EBV-associated cancers (Fig. 10). Autologous tumor-infiltrating lymphocyte therapy containing E6- and E7-specific T cells has shown efficacy against cervical and oropharyngeal cancer in a phase 2 study; however, it remains to be resolved if the clinical responses were associated with virus-specific or neoantigen-specific T cells in the products (301, 302). Subsequent studies have used autologous T cells transduced with a TCR specific for an HLA-A*02:01-restricted epitope encoded by E7 in patients with oropharyngeal and cervical cancer (303, 304). Patients demonstrated evidence of objective clinical responses. Given the lack of detectable HPV-specific T cells in the circulation of exposed healthy individuals (299), a repository of allogeneic VSTs is unlikely to be an option for HPV-associated diseases. A likely alternative allogeneic approach is the use of TCR-transgenic VSTs. Similar in approach to conventional allogeneic VSTs, this would employ a repository of T cells with diverse HLA coverage transduced with a panel of HPV-specific TCRs. The cost of viral vector manufacturing and testing is likely to prohibit their use for a transgenic T-cell repository that needs to provide diversity of HLA and antigen coverage. Instead, a nucleic acid-based approach would generate a more cost-effective VST repository.

HBV

Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). While currently available antiviral therapies are highly effective in controlling viral replication, a functional cure remains hugely challenging (305). Chronic HBV infection is primarily due to persistence of covalently closed circular DNA and impaired host immune control, which is associated with expression of PD-1 and reduced cytokine production by HBV-specific CD8⁺ T cells (306, 307). Different modalities of adoptive T-cell therapies have been explored for the treatment of chronic HBV infection and HCC. HBV-specific T cells, either expanded ex vivo from healthy virus-infected donors or patients, have shown clinical benefit. Adoptive transfer of HBV-specific T cells expanded from naturally infected donors or patients has shown clinical benefit, particularly in the post-transplant setting, leading to a reduction in viral recurrence and lower HBV surface antigen levels (308, 309). More recently, genetically engineered T cells expressing HBV-specific TCRs or CAR T cells have demonstrated potential therapeutic efficacy against HBV-infected hepatocytes in preclinical models (310–312). These TCR-transduced T cells are directed against HBV-encoded core or polymerase proteins. Early pilot studies have shown that TCR-transduced T cells can lead to regression of HBV-associated HCC (313, 314). Targeting of HBV surface antigens through CAR T cells has also been explored in clinical settings. However, there is a potential risk of hepatotoxicity due to on-target effects on HBV-infected hepatocytes. In addition, there are a number of other challenges that may limit the in vivo efficacy of adoptive T-cell therapies (313). These include limited long-term persistence of T cells in the immunosuppressive liver microenvironment and viral escape due to HBV’s genetic variability (315). Combination therapy with checkpoint inhibitors and targeting multiple HBV antigens has been proposed as an option to overcome in vivo challenges (316, 317). Early-phase clinical trials are ongoing, and while data remain limited, initial results suggest that adoptive T-cell therapy may contribute to viral suppression and tumor control in HBV-related liver cancer (318).

HIV

In spite of widespread use of combination antiretroviral therapy, HIV continues to be a global health challenge as these therapies fail to eliminate latent viral reservoirs. HIV-specific T-cell therapies have been explored as a therapeutic tool for the eradication of these reservoirs, which are predominantly composed of HIV-infected resting memory CD4⁺ T cells. Early clinical studies based on ex vivo-expanded autologous CD4⁺ T cells demonstrated safety and decreases in the percentage of CD4⁺CCR5⁺ cells in vivo (319). Subsequent studies by Hoffman and colleagues showed that adoptive transfer of HLA-matched T cells from HIV-negative twins in combination with highly active antiretroviral therapy led to an increase in CD4⁺ T-cell numbers in HIV-infected siblings (320). Ex vivo-expanded HIV-specific T-cell therapy has been assessed for clearance of persistent HIV infection (321). Adoptive immunotherapy with HIV-specific T cells was well tolerated, and an increase in CD8⁺ T-cell-mediated antiviral activity was observed in two of the six participants. However, this therapy did not result in a decline in the frequency of infected resting CD4⁺ T cells (321). More recent studies by Sohai and colleagues have assessed the therapeutic potential of HIV-specific T cells targeting conserved immunogenic protein regions of HIV Gag/Pol and HIV Nef (322). Adoptive immunotherapy with these T cells was generally safe, and an increase in HIV-specific T-cell and HIV anti-Env antibodies was observed in two participants. More importantly, some of the participants showed decreasing levels of intact proviruses and persistence of adoptive T-cell therapy-associated T-cell clones for ≥40 weeks post-infusions (322). Adoptive T-cell therapy in combination with the histone deacetylase inhibitor vorinostat (VOR) has been assessed for clearance of latent HIV infection (323). Six patients were treated with VOR and HIV-specific T-cell therapy, and some of these patients showed a limited decrease in quantitative viral outgrowth. However, this reduction in viral outgrowth was much lower than the sixfold threshold needed to definitively attribute decline to the study intervention. Further clinical studies with alternative combination therapies are required to achieve a meaningful outcome of reversing the HIV latency.

Varicella-zoster virus and herpes simplex virus

Herpesviruses such as Varicella-zoster virus (VZV) and herpes simplex virus (HSV-1 and HSV-2) establish lifelong latency following primary infection and can reactivate, particularly in immunocompromised HSCT and SOT recipients (324). HSV or VZV reactivation can result in severe or disseminated disease, including encephalitis, pneumonitis, and chronic mucocutaneous lesions (325). While standard antiviral agents such as acyclovir, valacyclovir, and foscarnet are effective in most cases, treatment-refractory or recurrent disease remains a challenge, particularly when drug resistance develops (326). There have been multiple studies demonstrating successful expansion of HSV and VZV-specific T cells from seropositive donors. These T cells are expanded using overlapping peptides from HSV gD, ICP4, and VZV gE and gB antigens (327, 328). Limited clinical reports have been published supporting the expansion and potential use of adoptive T-cell therapy for the treatment of HSV and VZV complications (329). Melzi and colleagues successfully treated chronic varicella with autologous VZV-specific T cells, which developed in a liver allograft (330). However, following adoptive immunotherapy, graft failure ensued and was probably due to massive cytolysis of infected hepatocytes. Similar preclinical experiences have been reported with HSV-specific T cells (331).

RESPIRATORY VIRUSES

While most research has focused on the development of VST therapy for viruses that establish persistent latent infections, transplant recipients are also at an increased risk of disease associated with respiratory viruses (RVIs). Predominant among these are rhinovirus, respiratory syncytial virus, AdV, coronaviruses (HCoV), parainfluenza, metapneumovirus, and influenza (332). Pediatric HSCT and SOT recipients have an elevated risk of complications from respiratory infection, with 14%–16% requiring hospitalization in the first year following transplant (333, 334). Respiratory infections are also more likely to progress to the lower respiratory tract in transplant recipients and are associated with late mortality in HSCT recipients. Reactivation of CMV following HSCT is a risk factor in the transmission of RVIs to the lower respiratory tract. This increased risk in transplant settings, and a lack of effective antiviral therapies for a number of RVIs, has led to the development of VST therapies for RVIs (335, 336).

Adenoviruses

The most advanced VSTs for RVIs are those developed against AdV (Fig. 7) (45, 52, 53, 84, 87, 91, 102, 103, 328, 337–339). Typically presenting as self-limiting common colds in most individuals, AdV infections are increasingly problematic in HSCT recipients (340). Pediatric recipients of allogeneic HSCT are particularly susceptible to AdV (341). Recent observations have shown an AdV infection incidence of 32% in the first 6 months following pediatric HSCT (342). While less frequent following adult HSCT, AdV infections still remain a significant risk. There are currently no approved antiviral drugs for AdV, although some common antivirals are used in first-line therapy (343). AdV are non-enveloped dsDNA viruses that encode 10 genes. CD4⁺ and CD8⁺ T-cell responses are generated against AdV (344), and both appear to play an important role in protection against disease (345). T cells specific for the capsid hexon protein predominate in response to AdV (346, 347) and have been the main target for VST approaches (77, 348). VST products for AdV are typically dominated by AdV-specific CD4⁺ T cells (63). Most clinical reports on the use of VSTs have employed T cells manufactured using a multi-virus-specific T-cell approach (77, 83, 85, 88, 90, 92, 95); however, single AdV-specific therapies have also been used (37, 91, 102, 338). A number of studies have also employed ex vivo enrichment using IFN-γ capture or MHC multimers as a source of AdV-specific VSTs (41, 45, 51–53, 103, 339). These approaches have all shown promise as platforms to prevent and treat AdV disease in early phase studies (Fig. 8); however, recent phase 3 studies aimed at preventing or treating AdV with posoleucel from AlloVir were abandoned due to likely failure to meet their primary endpoints.

Coronaviruses

HCoV are common post-transplant, with studies reporting that 17% of HSCT recipients develop a HCoV infection post-transplant, a third of which go on to develop lower respiratory tract infection (349). Infection in HSCT recipients can also lead to prolonged viral shedding. The COVID-19 pandemic brought into sharp focus the potential risk of HCoV infections in immunocompromised patients with no prior immunity (350).

It became apparent, not long after the start of the COVID-19 pandemic, that T-cell immunity plays an important role in the immune response to SARS-CoV-2. T-cell responses were quickly shown to be primed against most SARS-CoV-2-encoded antigens (351–353). While early evidence suggested that T cells contributed to pulmonary disease associated with severe COVID-19, more recent studies have shown that reduced antigen-specific T-cell immunity is associated with severe COVID-19 (354, 355). CD4⁺ and CD8⁺ T-cell responses are generated against SARS-CoV-2. Immunodominant CD4⁺ T-cell responses are directed toward the spike glycoprotein (spike), membrane protein, and the nucleocapsid (NCAP), while immunodominant CD8⁺ T-cell responses are preferentially induced against NCAP, ORF1a, ORF3a, and spike (351, 353). Prior to the development of vaccines for COVID-19, several groups with expertise in VST therapy developed SARS-CoV-2-specific VST strategies (64, 356–358). While the effective rollout of vaccines reduced the risk of severe COVID-19 in immunocompromised patients, an increased risk remains. The generation of VSTs to treat COVID-19 has relied upon allogeneic approaches using convalescent volunteers. This allogeneic approach has allowed the rapid administration of VST therapy in at-risk SARS-CoV-2-infected patients (Fig. 7). SARS-CoV-2-specific VSTs have been generated using overlapping peptide pools, primarily covering spike, or defined peptide epitopes covering the whole SARS-CoV-2 genome. Both approaches generate VSTs biased toward SARS-CoV-2-specific CD4⁺ T cells. Adoptive T-cell therapy with allogeneic VSTs for severe COVID-19 is feasible and well-tolerated in most recipients. Phase 1 and 2 studies have demonstrated clinical responses (Fig. 8) and improved recovery times compared to patients treated with standard of care (64, 359). Although less likely to be evaluated as a single virus-specific approach in pivotal studies due to the effectiveness of COVID-19 vaccines and antiviral drugs, these observations provide further evidence for the potential of VST therapy to treat acute RVIs.

IMMUNE MONITORING AND VST THERAPY

Reconstitution of robust cellular immunity has been recognized as a critical player in protecting transplant recipients from virus-associated complications. In vitro diagnostic cellular immunity assays are widely used to assess the functional and phenotypic profile of lymphocyte subsets, including virus-specific CD4⁺ and CD8⁺ T cells, natural killer (NK) cells, B cells, and regulatory T cells. Immune monitoring assays have been extensively validated and are now available as commercial diagnostic kits. These include QuantiFERON CMV (QIAGEN, Hilden, Germany) (360–363), T-SPOT.CMV (Revvity, Waltham, MA) (360, 364), T-Track CMV (Mikrogen, Neuried, Germany) (361, 365), and CMV inSIGHT T Cell Immunity Panel (Eurofins Viracor, Kansas City, KS) (366). These commercial assays are designed to assess virus-specific T-cell immunity in either whole blood or purified PBMC following stimulation with synthetic peptides. These assays have been used for adapting immunosuppressive therapy and anti-viral prophylaxis according to the risk of infection for transplant recipients. Furthermore, assessment of the frequency and function of virus-specific T cells in HSCT and SOT recipients can be used to stratify risk and guide preemptive therapies (367–370). Indeed, clinical studies have shown that the absence of detectable CMV-specific CD8⁺ T cells in the early post-transplant period is associated with increased risk of CMV reactivation and progression to CMV disease (371). While ex vivo immune monitoring has been successfully used for viruses like CMV, application of these assays for other viruses, such as BKPyV, JCPyV, and human herpesvirus 6, can be technically challenging due to low precursor frequency and subdominant immunogenicity of viral antigens. To address this limitation, newer technologies such as immunophenotyping and high-throughput TCR sequencing are being evaluated to provide sensitive and scalable solutions for virus-specific T-cell monitoring (62, 93, 372). Antiviral immune monitoring can be used both before and after VST adoptive immunotherapy. Pre-infusion testing ensures that donor-derived VSTs recognize the relevant viral epitopes presented in the patient’s HLA context. Post-infusion monitoring assesses in vivo expansion, persistence, and antiviral efficacy of the infused cells. Studies have demonstrated that successful expansion of EBV- or CMV-specific T cells post-VST therapy correlates with viral clearance and clinical response, with minimal risk of GvHD. Deep sequencing of TCRs before and after CMV-specific adoptive T-cell therapy has demonstrated that immune control of CMV in SOT recipients is coincident with significant repertoire remodeling and is often absent in patients who fail to respond to adoptive T-cell therapy (373). It will be important to use this knowledge to establish validated cut-off thresholds and integrate antiviral immune monitoring into clinical management of patients following VST adoptive immunotherapy.

EMERGING INNOVATION AND TECHNOLOGIES FOR VST MANUFACTURING AND CLINICAL ACCESS

As VST therapies progress through early-stage development and clinical trials, the challenge of efficient, scalable, and accessible manufacturing, and rapid and equitable clinical access becomes increasingly pressing. Over the last few years, academic institutions and the biopharmaceutical industry have invested considerable resources to address these challenges. Approval of the allogeneic EBV-specific T-cell therapy EBVALLO by the European Medicines Agency has provided a new impetus to progress these therapies to a broader range of viral complications (81). Unfortunately, the recent failure of phase 3 clinical trials of an allogeneic multi-virus-specific T-cell therapy (posoleucel) targeting AdV, BKpyV, CMV, EBV, HHV-6, and JCPyV has dampened the interest in the clinical development of VST therapies. Another clinical trial, testing allogeneic EBV-specific T-cell therapy ATA188 in MS patients, also failed to achieve its clinical objective (189). While the precise reasons for these failures are unknown, it is possible that the VST products used in these clinical trials may not have appropriate potency and specificity to achieve the intended outcome. Furthermore, treatment of patients with late-stage end-organ disease and high viral load could also lead to poor clinical responses. Adoptive immunotherapy with VSTs at an earlier stage of viral complication offers much better potential for clinical response, and switching of allogeneic VST products with different HLA restrictions should also be considered to improve therapeutic outcome (78).

There are now emerging technologies, particularly artificial intelligence and machine learning, in refining manufacturing processes, and these may offer opportunities to improve VST potency. These technologies can also offer potential improvements in consistency, quality assurance, and real-time process control. Advanced sensors and automation platforms have been employed as enablers of predictive modeling and optimization in cell therapy production. Furthermore, decentralized models enable therapy production closer to the point of care, reducing logistical complexity and improving patient access. These strategies also offer resilience in the face of supply chain disruptions and may promote more equitable therapy access across all regions (including remote and regional locations). There is also the need for risk-based, adaptive regulatory frameworks that balance innovation with safety and efficacy. Engagement between developers and regulators will help to foster the validation and implementation of novel manufacturing tools, including real-time analytics and process automation. Partnerships among academic institutions, industry stakeholders, regulatory bodies, and patient advocacy organizations are essential for harmonizing standards, accelerating innovation, and ensuring patient-centered development. By integrating emerging technologies, rethinking manufacturing models, and evolving regulatory paradigms, VST therapies can continue to advance toward scalable, accessible, and life-saving therapies for transplant recipients with virus-associated complications.

CONCLUSION

Unlike an antiviral drug or monoclonal antibody, VST therapies are inherently complex, comprising hundreds to thousands of distinct T cells within a product and very little overlap between T cells in different products. However, it is this complexity that likely provides VST therapy an advantage over antiviral drugs, limiting the likelihood of resistance due to escape and more closely mirroring natural virus-specific T-cell responses that are primed endogenously following infection. The limitations of recent allogeneic VST approaches are more likely a consequence of a lack of a clear understanding of the key potency attributes associated with efficacy. Current omics approaches to define these potency attributes should enhance our understanding of VST efficacy and lead to better product uniformity and qualitative improvements in VST therapies.

Biographies

Corey Smith, Associate Professor, completed his Ph.D. studies in 2004 at the University of Melbourne and then took up a research position at the QIMR Berghofer Medical Research Institute. He is currently appointed as Head of Translational and Human Immunology. Assoc. Prof. Smith is also appointed as Honorary Associate Professor at the University of Queensland and Queensland University of Technology, Brisbane, Australia. His work focuses on the development of immunotherapeutic approaches to treat diseases and cancers associated with persistent viral infections. This has led to the initiation of a number of adoptive immunotherapy studies. His work has also focused on understanding the mechanisms that influence the efficient induction of T-cell responses to persistent human viral infections as well as the role that immune evasion strategies play in virus-associated cancer and disease.

Rajiv Khanna, Professor, obtained his doctorate from India and undertook his post-doctoral training at the Queensland Institute of Medical Research (QIMR), Brisbane, Australia. He is the Co-Director of Queensland Immunology Research Centre and is currently appointed as a Distinguished Scientist at QIMR Berghofer Medical Research Institute. Prof. Khanna is also appointed Honorary Professor at the University of Queensland and Griffith University, Brisbane, Australia. Prof. Khanna is a Fellow of the Australian Academy of Health and Medical Sciences and was awarded the Order of Australia in 2017. He has extensive expertise in immunotherapy clinical trials, herpesvirus immunology, and vaccine development. Over the last two decades, his group has successfully translated his research toward the development of novel T-cell-based immunotherapeutic strategies for the treatment of transplant recipients and autoimmune diseases. Prof. Khanna has been invited by various national and International organizations for expert advice on developing clinical guidelines and scientific review.