Polyomavirus-associated nephropathy is a pertinent cause of poor renal allograft survival. Absence of defensive immunity toward BK polyomavirus may favor the occurrence of BK polyomavirus-active infection and influence the progression to polyomavirus-associated nephropathy. Humoral immune responses may offer incomplete protection. In this review, available data on both humoral and cellular immunity were examined, with a con­centration on BK polyomavirus-specific T cells; in addition, their roles in BK polyomavirus cellular immune response and immunotherapy were discussed. This traditional narrative review used PubMed and Medline searches for English language reports on BK polyomavirus immune response and BK-specific T cells published between January 1990 and November 2017. The search included the key words BK virus, BK polyomavirus, immune and response, and specific T cells. Monitoring BK polyomavirus-specific T cells has both therapeutic and prognostic value. Innovative cellular immunotherapy approaches, including deve­lopment of vaccinations and infectious recombinant BK polyomavirus, could further contribute to the prevention of BK polyomavirus infection and related diseases.

Key words : Cellular immunity, Cytotoxic T lymphocytes, Humoral immunity, Kidney, Renal transplantation

Introduction

The BK polyomavirus (BKPyV) is a small (~5 kb), nonenveloped virus with an icosahedral capsid and a core of circular double-stranded DNA that encodes for the early nonstructural genes encoding large T and small t antigens; the late genes then encode the capsid proteins (VP1, VP2, and VP3) and the agnoprotein and a noncoding control region, which protect viral promoters and the origin of replication.¹

BK virus is currently the most challenging infectious reason for renal allograft dysfunction and graft loss.² Polyomavirus-associated nephropathy (PVAN) has been shown to be the cause of graft loss in 10% to > 80% of cases.^3,4 The imbalance between BKPyV replication and cellular immunity has been reported as the main risk factor for the development of severe BKPyV infection/PVAN.^5,6 Comoli and associates postulated that BKPyV-specific T cells play a protective role in development of and recovery from PVAN.⁶ In this review, the available data on both humoral and cellular immunity are presented, focusing on the BKPyV-specific T cells; in addition, their roles in BKPyV cellular immune response and cellular immunotherapy are discussed.

To focus on the characteristics of the immune response to BKPyV with a focus on BKPyV-specific T cells, this traditional narrative literature review used the PubMed and Medline search engines to identify relevant reports published between January 1990 and November 2017 using the key words BK virus, BKPyV, immune and response, and specific T cells. Ninety articles were identified, with abstract-only and non-English articles excluded. The fundamental purpose of this review was to create a readable synthesis of the best resources available in the literature for this important topic. Immune responses (innate and humoral) to BKPyV and the effects of immunosuppression on these responses were included as best as possible. Moreover, monitoring of BKPyV-specific T-cell immunity and the use of BKPyV-specific immune response as therapeutic strategy were also included.

Innate Immunity and BK Virus
The innate immunity system consists of different cell types, including monocytes (macrophages in the tissues), dendritic cells (DCs), natural killer (NK) cells, eosinophils, basophils, neutrophils, and mast cells, as well as soluble molecules, including those in the complementary system, pentraxins, lectins, ficolins, and some nonimmunologic molecules participating in inhibitory functions against microbes such as transferrin and lactoferrin.⁷

Innate immunity cells suppress virus replication, activate adaptive immunity to eradicate infection, and play a main role in the development of PVAN through either direct lysis of the infected target cells and losing their function or release of the allograft antigens through lysis of the infected cells and activation of the adaptive immune system.⁷

Natural killer cells identify virally infected cells through down-regulation of class I major histocom­patibility complexes (MHCs) and inhibitory receptors and through up-regulation of activator molecules like UL16-binding (ULB) protein, MHC class I polypeptide-related sequence A, and MHC class I polypeptide-related sequence B and through up-regulation of activating receptors on the infected cells. Natural killer cells then kill or induce apoptosis in virally infected cells after activation.⁸ Bauman and associates stated that BKPyV inhibits NK cell acti­vation through suppression of the NKG2D/ULBP3 interaction.⁹ Therefore, patients with PVAN have low numbers of NK cell immunoglobulin-like receptors (KIRs) as activating receptors and KIR3DS1 compared with kidney recipients with stable function and no PVAN.¹⁰

Furthermore, DCs and macrophages (antigen-presenting cells) can modulate the disease in an indirect manner by presenting BKPyV antigens and also by producing various cytokines.¹¹ Accordingly, several studies have used DCs as vehicles for direct and indirect immunotherapy against viruses, including BKPyV, in patients with immunode­ficiency.¹² Furthermore, the absolute number of DCs in PVAN patients is reduced compared with numbers shown in other kidney recipients, despite the presence of ureteral stent and the use of tacrolimus.¹³ Nonetheless, the DC level and function could be further studied as biomarkers for the prediction of BKPyV reactivation and disease.

With regard to monocytes, patients with BKPyV viruria (not PVAN) have increased levels of soluble interleukin (IL)-1 receptor antagonists in their urine, a counter-regulator of monocyte activation that can be produced by monocytes.¹⁴ Geetha and associates reported that BK virus reactivation is seen more in renal transplant patients with antineutrophil cytoplasmic antibody-associated vasculitis who have neutrophil dysfunction than in renal transplant patients with normal neutrophils.¹⁵ The presence of tubulitis in association with eosinophil infiltration is the main pathologic feature of PVAN,¹⁶ and eosinophilic enzymes may induce some pathologic events during PVAN.

Soluble molecules of innate immunity participate in viral elimination by either directly targeting the viruses, like the complementary system, or indirectly by opsonization of the particle.¹⁷ Collectively, the complementary system may not only disrupt the function of transplanted kidney cells but may also accelerate PVAN through induction of inflammation by complementary system anaphylatoxins like C3a and C5a, which has been reported in several com­plementary system-dependent hypersensitivities.¹⁸

Lectins, especially mannose-binding lectin, can recognize viral carbohydrates and activate innate immunity by activation of the complementary system and also opsonize the viral particles.¹⁹

Innate immunity cytokines target either innate or adaptive immunity cells. The most important innate immunity cytokines are tumor necrosis factor α (TNF-α), IL-1, IL-6, IL-8, IL-10, IL-12, IL-15, and IL-18.20 Corallini and associates revealed that BK virus transformed endothelial cells to express transforming growth factor β mRNA more than normal murine endothelial cells; therefore, the cytokine may induce lysis of infected cells by cytotoxic T lymphocytes (CTLs) indirectly.²¹

Another study revealed that interferon-gamma (IFN-γ) inhibits BKPyV reactivation through up-regulation of IL-12 in a positive feedback.²² Kariminik and associates⁷ mentioned that new treatment strategies such as depletion of antineutrophil cytoplasmic antibody, up-regulation of ULBP3, and use of anti-transforming growth factor β can be considered for the treatment of BKPyV reactivation. In addition, use of receptor antagonists, like TLR3, can be considered as future treatment strategies. Moreover, targeting proinflammatory molecules such as the use of micro-RNAs may be applicable to the treatment of PVAN.⁷

Effects of Immunosuppression and Transplant Surgery on Innate Immunity
Immunosuppressive therapy inhibits NK function; specifically, cyclosporine A affects NK cell function, phenotype,²³ and proliferation.²⁴ Furthermore, prednisolone inhibits their proliferation when exposed to allogenic tubular epithelial cells, and tacrolimus may counter their capacity to degranulate in the same context. Mycophenolate mofetil possibly inhibits proliferation induced by IL-2.²⁴ Hackstein and associates demonstrated that levels of all DC subtypes were lower in kidney transplant recipients treated with long-term immunosuppression (more than 1 year) than in age- and sex-matched controls, independently of total leukocyte count.^24-25

Transplant surgery itself induces a strong decline in the number of DCs (and possibly with a greater reduction in plasmatoid DC 103) in kidney transplant recipients and in kidney donors. This decline can last up to 3 months postoperatively. As opposed to donors, patients on chronic immunosuppression fail to recover to normal counts.²⁶

BK Humoral Immunity
It is controversial as to whether humoral immunity is involved in control of BK and whether intravenous immunoglobulin (IVIG) therapy could have a role in its treatment. Although BKPyV-seropositive reci­pients are not protected from viral replication and PVAN,2 BK virus-seronegative recipients are at higher risk of viral replication and nephropathy.^7-11 Moreover, recipients who receive kidneys from donors with high titers are more likely to develop viruria.9 Several studies have reported that high antibody titers in recipients do not correlate with protection from PVAN.^10,11

BK Cellular Immunity
BK polyomavirus-specific T cells
The key role of T-cell immunity in the control of BKPyV infection was initially suggested by the indirect evidence of increased incidence of reactivation and clinical disease related to the degree of immune suppression.²⁷

Schachtner and colleagues found that rapid development of BKPyV-specific T cells without therapeutic interventions is associated with self-limited BKPyV reactivation.²⁸ Moreover, it was established that CTLs are important for the clearance of acute polyomavirus infection in a mouse model.²⁹

Comoli and associates reported that patients with BKPyV-active infection and good renal function had a mean BKPyV-specific lymphocyte frequency 2 logs lower than healthy controls and in the same range as BKPyV-seropositive recipients without active infection. Moreover, patients with PVAN showed undetectable levels of BKPyV-specific cells.⁶ Comoli and associates suggested that a BKPyV immunity analysis, as predictor of risk and response to treatment of PVAN, and the identification of patients at a preclinical stage could further improve disease outcomes. Moreover, they stated that progression to PVAN can be safely prevented if BKPyV viremia is used to guide therapeutic intervention.³⁰

Among patients with positive viremia (or even high positive viruria), assessment of BKPyV-specific T-cell frequency could allow identification of those more likely to progress to PVAN. In this regard, it has been recently shown that the presence of large T antigen-specific, but not VP1-specific, secreting cytokines and cytotoxic T cells at an early time after transplant protect patients from risk of BKPyV viruria. In addition, among patients with BKPyV viruria, the ability to mount BKPyV large T antigen-specific immunity is associated with protection from development of BKPyV viremia.³¹ Future studies should combine virologic and immunologic treatment.

The control of BKPyV replication is correlated with differentiated expansion of BKPyV-specific cellular immune responses.32 Binggeli and associates compared the frequency of IFN-γ-secreting peri­pheral blood mononuclear cells (PBMCs) after stimulation with overlapping peptide pools covering BKPyV large T antigen and VP1 capsid proteins using enzyme-linked immunospot (ELISPOT) assays.³³ In kidney transplant recipients with decreasing or past plasma BK virus loads, significantly higher median BKPyV-specific IFN-γ responses were detected compared with kidney transplant recipients with increasing or persisting BKPyV loads (large T antigen level of 78 spot-forming units [SFU; range, 8-524] vs 22 SFU [range, 0-120]; P = .003; VP1 level of 285 SFU [range, 45-1432] vs 53 SFU [range, 0-423]; P = .001).³² Moreover, the recovery of cellular immune response to large T antigen corresponds with resolution of active BKPyV infection; this could be useful in monitoring cellular immunity in patients after treatment with reduction in immunosuppressive therapy.²

BK polyomavirus-specific protein response
Mueller and associates reported that that all 5 BKPyV-specific proteins were able to provoke memory T-cell responses, which were dominated by CD4 T cells.4 Zhou and associates investigated T-cell responses to overlapping peptide mixtures corresponding to the whole BKPyV major T antigen and the major capsid protein (VP1) in PBMC samples in a cohort of healthy BK virus-seropositive individuals.34 Most of the BKPyV-specific CD4 T cells, in addition to expressing CD40L (CD154) and being IFN-γ, IL-2, and TNF triple cytokine producers, contained both granzyme A and granzyme B and degranulated/mobilized CD107 in response to antigen-specific stimulation. Therefore, these T cells represent potentially functional BK virus-specific cytotoxic CD4-positive T lymphocytes that may play a part in maintaining persistent memory T-cell responses to the virus and thus contribute to the immune control of BKPyV in healthy individuals.^4,34,35

Large T antigen response
The main multifunctional regulatory protein anti-BKLTD1 represents a serologic marker of extensive BKPyV exposure.^36,37 Specific IFN-γ-producing T cells have been shown to be more likely CD8 positive.^38-39 The development of large T antigen-specific T cells is related to recovery from BK virus reactivation.³⁵ Failure to mount large T antigen-specific T cells predisposes to BKPyV reactivation.³⁰ Positive immunohistochemistry for intranuclear large T antigen is the standard confirmatory assay for the diagnosis of PVAN in renal allograft biopsies.³⁷

VP1 antigen and BK virus-like particle response
VP1 antigen response is most sensitive and an early serologic marker of BKPyV exposure.^28,37 Specific IFN-γ producing T cells are more likely to be CD4 positive.⁴³⁸ Persistent BKPyV viruria and viremia were associated with significantly higher anti-BK virus-like particle responses, which remained elevated for prolonged times after transplant.

VP3 antigen response
The VP3 antigen has the highest incidence of BKPyV-specific memory T-cell responses. It is the only antigen inducing memory T-cell responses in all patients with severe PVAN.⁴

BK Polyomavirus-Specific T-Cell Kinetics
Patients with PVAN showed the highest frequencies of BKPyV-specific T cells at recovery, the highest increase in BKPyV-specific IgG, and persistence of increased IgM levels (P < .05).²⁸ Although kidney transplant recipients with self-limited BKPyV reactivation were able to show BKPyV-specific cellular immunity within a period of 1 month after diagnosis of BKPyV reactivation without reduction of immunosuppression, kidney transplant recipients with PVAN only developed VP1-, VP2-, and VP3-specific T cells at a median period of 5 months and only after therapeutic interventions, with cleared BKPyV reactivation (developed small T- and large T antigen-specific T cells) after a median period of 8 months.²⁸ Schachtner and associates suggested using these prognostic values of BKPyV-specific immune monitoring to identify those patients at risk of PVAN and to aid in the management of therapeutic interventions.²⁸

BK Virus-Specific T-Cell Response and Dendritic Cells
Dendritic cells are effective antigen-presenting cells central for the induction of antiviral cytotoxic T-cell responses.⁴⁰ The deficiency, before and after transplant, of peripheral blood DCs in patients with biopsy-proven PVAN predisposed patients to BKPyV reactivation.⁵ Womer and associates postulated that pretransplant peripheral blood DC is a sensible predictor for the development of posttransplant viremia. They identified that a pretransplant total DC level of 23 483 cells/mL is the best cutoff value for predicting posttransplant viremia with a sensitivity of 87% and specificity of 46%.⁵

BK-Specific T Cells and HLA Mismatch
Several studies have shown a trend for an increased incidence of PVAN in kidney transplant recipients of HLA class I-mismatched allografts that might impair the immune response against BKPyV.^2,39,41 It is not clear whether CTLs are able to identify viral epitopes presented by nonself class I alleles in the allograft and efficiently destroy infected cells or whether this response was elicited by classical antigen pre­sentation in tissues of the recipient or via cross-recognition of BKPyV VP1p44 and VP1p108 CTLs epitopes on other class I molecules in the infected graft.

Han Lee and associates found that polyomavirus preferentially replicates in the allogeneic kidney grafts, accelerating graft failure in an animal model. In addition, acute polyomavirus infection of mouse allograft recipients augmented the alloreactive CD8-positive T-cell response, while maintaining the anti-viral CD8-positive T-cell response. Moreover, these findings demonstrate a potential role for an enhanced anti-donor T-cell response in the pathogenesis of PVAN.⁴²

Chen and associates have identified two HLA-A*0201-restricted 9-amino acid CTL epitopes of the BKPyV major capsid protein VP1, VP1(p44), and VP1(p108). These epitopes were recognized by CTLs in 8 of 10 (VP1[p44]) and 5 of 10 (VP1[p108]) HLA-A*0201-positive healthy individuals, whereas both epitopes elicited a CTL response in 10 of 10 kidney transplant recipients with biopsy-proven PVAN, although at variable levels. After in vitro stimulation with the respective peptides, CTLs directed against VP1(p44) were more abundant than against VP1(p108) in most healthy individuals, whereas the converse was true in kidney transplant recipients with PVAN, suggesting a shift in epitope immuno­dominance in the setting of active BKPyV infection. A strong CTL response in kidney transplant recipients with PVAN appeared to be associated with decreased BKPyV viral load in blood and urine and low anti-BKPyV antibody titers, whereas a low or undetectable CTL response was correlated with viral persistence and high anti-BKPyV antibody titers. These results suggest that this cellular immune response is present in most BKPyV-seropositive healthy individuals and plays an important role in the containment of BKPyV in kidney transplant recipients with PVAN. Interestingly, the BKPyV CTL epitopes bear striking homology with the recently described CTL epitopes of the other human polyomavirus, including JC virus, JC virus VP1 (p36), and VP1 (p100).³⁶

BK Polyomavirus-Specific T-Cell Response and Immunosuppression
Reducing immunosuppression is associated with increased BKPyV-specific cellular immune responses, decline in BKPyV loads, and emergence of BKPyV-induced IFN-γ secreting cells to reach the same or higher levels as found in healthy controls.³⁰ Therefore, low-dose tacrolimus may be a successful strategy to prevent BK virus replication and disease in kidney transplant patients.⁴³ Araki and associates⁴⁴ found that mTOR inhibition may increase antiviral memory CD8-T-cell differentiation. Egli and associates45 found that, in kidney transplant recipients, BKPyV-specific T-cell responses were inversely correlated with tacrolimus trough levels (R2 = 0.28, P = .002) but not with mycophenolate levels, prednisone, or overall immunosuppressive dosing. They found that recipients with tacrolimus trough levels below 6 ng/mL had significantly higher BKPyV large T-antigen-specific activity compared with patients with tacrolimus above 6 ng/mL. Therefore, they supported the present recom­mendation for treating BKPyV by reducing tacrolimus below 6 ng/mL and cyclosporine below 150 ng/mL and suggested that the lower targets be 3 and 100 ng/mL as first steps, whereas conversion to mTOR inhibitors may be a second step.⁴⁵ Prednisolone and calcineurin inhibitors impair cytokine secretion and cytotoxic capacity of BKPyV-specific T cells, whereas rapamycin does not. BKPyV-specific T cells can be detected and categorized by CD137, CD154, and granzyme B expression.⁴⁶

Monitoring of BK Polyomavirus-Specific T-Cell Immunity
Monitoring the BKPyV-specific immune response might prove useful in guiding therapeutic interventions.⁴⁷ Evaluation of virus-specific T-cell responses can be made by different methods, including intracellular cytokine staining (ICS), MHC multimer-based assay, and ELISPOT assay.⁴⁸ In addition to these virus-specific assays, the ImmuKnow assay is a nonspecific test used to evaluate the overall CD4-positive immune response.

Intracellular cytokine staining
Intracellular cytokine staining is used to assess the impact of decreased immunosuppression on BK virus immunity and to clarify the role of specific T cells in the pathogenesis of PVAN.⁴⁹ Most studies have analyzed virus-specific T-cell responses using ICS for IFN-γ using flow cytometry. Moreover, ICS allows for evaluation of polyfunctionality of T cells. Whole blood or isolated PBMCs are stimulated with virus-specific peptides or viral lysates.⁴⁸ It is a fast method that needs low-volume blood; however, it requires a flow cytometer, is HLA independent, and is not standardized.⁴⁸

Major histocompatibility complex multimer staining
The MHC method evaluates CD8-positive T-cell responses; however, it is epitope specific and requires knowledge of the patient’s HLA type.⁴⁸ Major histocompatibility complex multimer-based assays are characterized by the direct staining of peptide-specific T cells using peptide-conjugated MHC class I tetramers or pentamers. This is not possible for HLA-class II (CD4-positive T-cells) availability of tetramers for various HLAs. This fast method needs low-volume blood; however, it requires flow cytometer and is not standardized.⁴⁸

Interferon-gamma enzyme-linked immunospot assay
The ELISPOT assay determines the number of T cells secreting IFN-γ in specific response to a viral agent. After stimulation with viral peptides or viral lysates, the produced IFN-γ is captured by a specific antibody and then quantified using a labeled antibody. This assay evaluates both CD4-positive and CD8-positive responses.⁴⁸

This fast and sensitive test² is HLA independent and has the same sensitivity and requires the same amount of blood as flow cytometry. It is easier to perform than flow cytometry. It is time and resource consuming because DCs have to be obtained and expanded for each patient.⁶ However, it allows analysis of only 1 marker. No data on phenotypic and functional characteristics of antigen-specific T-cells are possible (eg, CD4 or CD8, other cytokines, cytotoxicity, and developmental stage).

ImmuKnow assay
The Cylex ImmuKnow assay (Cylex Inc., Columbia, MD, USA) is a commercially available nonspecific assay. It measures the overall immune response and is indicative of immunosuppression. It determines the amount of ATP produced in response to whole blood stimulation by a specific mitogen (phytohemagglutinin).⁴⁸

Other monitoring strategies
Weist and associates⁴⁶ used TNF-receptor family members 19 to 23 and the effector molecule granzyme B for monitoring. The expression of these markers is significantly less affected by immuno­suppressive drug treatment than the expression of effector cytokines. Accordingly, the sensitivity to detect BKPyV-specific T cells is improved compared with the standard IFN-γ-based method and can provide details on the phenotype and functional characteristics of these cells in the context of clearance of BKPyV in immunocompromised kidney transplant patients.⁴⁶

BK Polyomavirus-Specific Immune Response as Therapeutic Strategy
Although both humoral and cellular immunity responses may be essential, BK virus-specific T-cell immunity appears to play a pivotal role in con­trolling BKPyV replication.^6,38,47,50 Several studies have worked to develop cytotoxic cytokines, BKPyV-specific CTLs, MHC-unrestricted cytotoxic popu­lations, JC-specific T cells, vaccinations, and recombinant BKPyV.

BK polyomavirus-specific cytotoxic T lymphocytes and cytotoxic cytokines
Expanded CD4 and CD8 cells have been shown to respond to restimulation with BKPyV peptides principally from VP1, large T, or small t antigens; produce multiple cytokines; and display cytotoxic activity against antigen-coated targets.⁵⁰

Rapid restoration of virus-specific T immunity via adoptive transfer of ex vivo-generated T cells has been proven to be a powerful therapy for patients with advanced cancers and refractory viral infections such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV).⁵¹ Riddell and associates demonstrated that virus-specific T-cell clones from a healthy donor could be generated ex vivo from autologous CMV-infected fibroblasts.⁵² Several subsequent trials of adoptive immunotherapy with virus-specific T cells derived from the transplant donor have established their safety and potency, both for prevention and for treatment of CMV disease. Application of virus-specific T cells has been subsequently expanded, first to the generation of EBV-specific T cells^53,54 and then to the generation of multivirus-specific T cells targeting common posttransplant viral pathogens, including adenovirus, BKPyV, and human herpes virus 6 (HHV-6).^55-57 Interestingly, a third group of BKPyV-specific T cells generated from a wide array of BKPyV peptides was used to successfully treat 10 patients with BKPyV hemorrhagic cystitis after allogeneic stem cell transplant. These BKPyV-specific T cells have yet to be reported in PVAN after solid-organ transplant.⁵⁶

Generation of BKPyV-specific T cells
For clinical use in adoptive immunotherapy, BKPyV-specific T cells were generated using monocyte-derived DCs pulsed with overlapping peptide mixes spanning the 5 BKPyV proteins VP1, VP2, VP3, large T antigen, and small t antigen.⁵⁰ Furthermore, Comoli and associates provided an alternate method of BKPyV -specific CTL generation based on the stimulation of PBMCs with DCs pulsed with inactivated BKPyV, in the presence of IL-7 and IL-12, 2 cytokines that have been demonstrated to activate DCs, enhance T-cell cytotoxicity, and promote CD8þ CTL survival.^6,58

Moreover, Lamarche and associates developed a Clinical-Scale Rapid Autologous BKPyV-specific T cell with stimulated PBMCs from healthy controls and viremic kidney transplant recipients using BKPyV peptide libraries loaded or not on monocyte-derived DCs. They found that autologous BKPyV-specific T-cell lines can be reliably generated from viremic kidney transplant recipients.²⁴

More recently, techniques have been developed to utilize MHC multimers,⁵⁹ or IFN-capture technology to allow rapid selection and enrichment of virus-specific T cells.60 Multimer technology and magnetic beads are available to produce untouched T cells in a single-step, good manufacturing practice-compliant procedure.⁵⁹

For the successful generation of virus-specific T cells, it is necessary to identify the immunogenic antigens of the virus. The immunodominant epitopes of herpes viruses, such as CMV and EBV, have been well defined, enabling researchers to successfully expand CMV- or EBV-specific T cells by using synthetic viral peptides or overlapping peptide pools.^61,62

Multivirus-specific cytotoxic T lymphocytes
Gerdemann and associates⁶³ described a system that rapidly generates a single preparation of polyclonal (CD4-positive and CD8-positive) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, adenovirus, BKPyV, HHV-6, respiratory syncytial virus, and influenza) that commonly cause posttransplant morbidity and mortality. Cytotoxic T lymphocytes can be rapidly produced (in 10 days) by a single stimulation of donor PBMCs with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines IL-4 and IL-7.⁶³

Tzannou and associates⁵⁵ generated a bank of virus-specific T cells that recognized 5 common viral pathogens: EBV, adenovirus, CMV, BKPyV, and HHV-6. The virus-specific T cells were administered to 38 patients with 45 infections in a phase II clinical trial. A single infusion produced a cumulative complete or partial response rate of 92% (95% confidence interval, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKPyV (n = 16), 94% for CMV (n = 17), 71% for adenovirus (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). The use of banked virus-specific T cells is a feasible, safe, and effective approach to treat severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT), including infections from two viruses (BKPyV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the virus-specific T cells ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.^55,57,64 Another exciting aspect of T-cell therapy against polyomaviruses is the fact that both JC virus and BKPyV can be targeted simultaneously because of their high sequence homology.⁶⁵

Major histocompatibility complex-unrestricted cytotoxic populations
Numerous studies have used MHC-unrestricted cytotoxic populations, particularly CD3+/TCRγδ+ cells, to control BKPyV reactivation.^6,66 Production of inactivated virions under good laboratory practice conditions have allowed reactivation of T-cell lines with multiple specificities to be used for patients of any HLA type.^30,50 Unfortunately, an HLA-restricted T-cell response to BKPyV is so far poorly characterized and only few VP1 and large T-antigen epitopes have been defined, with most of the described epitopes being HLA A*02 restricted.⁶⁷

JC virus-specific T cells
Several studies have generated BKPyV-specific T cells for possible use in adoptive cell transfer by antigenic stimulation with 15-mer peptide sequences derived from VP1, VP2, VP3, small t antigens, and large T antigens.^66,68 Balduzzi and associates successfully treated HSCT recipients affected by progressive multifocal leukoencephalopathy with JC polyomavirus-specific T cells obtained by stimulating PBMCs from the HSCT donor with a pool of 15-mer peptides spanning the whole sequence of JC virus VP1 and the large T antigen.⁶⁹ Delbue and associates followed a similar approach with JC virus-mediated PVAN.⁶⁸

Vaccinations
During their vaccine development, because peptide vaccination had been generally a poor inducer of cellular immunity, Caparrós-Wanderley and associates⁷⁰ used virus-like particles (VLPs) as optimal carriers of antigen delivery. They analyzed the stability and immunogenicity of murine polyomavirus VLPs after intranasal administration without adjuvant. No morphologic or immunologic changes were noticed in a preparation of these VLPs stored for 9 weeks at room temperature. Strong humoral and cellular (Th1) responses were obtained after a single 5.55-μg immunization, with responses efficiently boosted after a second dose. However, at dose concentrations above 0.22 μg/μL, these VLPs appeared to aggregate; when used for immuni­zations, they failed to induce a strong cellular response, although the humoral response was not affected.⁷⁰

Recombinant BK Polyomavirus
Husseiny and Lacey⁷¹ developed a novel approach of engineering recombinant BKPyV by fusing sequences coding for foreign polypeptides via the self-processing 2A peptide in frame to the BKPyV agnoprotein or VP2 capsid protein genes to minimize alterations to native BKPyV polypeptide sequences and expression. They used this process to potentially allow preservation of viral viability. An infectious recombinant BKPyV, in which the large T sequence could be adapted to elude any safety implication related to the potential tumorigenicity of large T antigen, was recently proposed as an alternative strategy for vaccination in the context of BKPyV.⁷¹

Role of Intravenous Immunoglobulin
Intravenous immunoglobulin preparations contain BKPyV-neutralizing antibodies that can bind several human viruses including BKPyV.⁷² In addition, IVIG can exert a beneficial effect, including interference of receptor binding, producing a conformational change in the bound protein, complement-dependent cyto­toxicity, viral agglutination, and antibody-facilitated phagocytosis.73 Surprisingly, an increase in the plasma BKPyV viral load was shown in a PVAN patient after IVIG therapy.⁷⁴ Sharma and associates suggested that patients with persistent viremia unresponsive to immunosuppression reduc­tion and patients with histologically established BKPyV nephritis should be considered for IVIG therapy.⁷⁵

Conclusions

BK polyomavirus-specific T cells are the best indicator of response to therapeutic intervention in viremic patients. Detection of BKPyV-specific T cells allows for a cautious modulation of immune suppression. In addition, it has a prognostic value by identifying those patients at risk of PVAN and by aiding in the management of therapeutic interventions. New immune-based strategies, including vaccination and cell therapy, could further contribute to the pre­vention of BKPyV infection and related diseases.

References:

1. 1. Bohl DL, Brennan DC. BK virus nephropathy and kidney transplantation. Clin J Am Soc Nephrol. 2007;2 Suppl 1:S36-46.
   CrossRef - PubMed
   
2. Hirsch HH, Knowles W, Dickenmann M, et al. Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med. 2002;347(7):488-496.
   CrossRef - PubMed
   
3. Comoli P, Ginevri F. Monitoring and managing viral infections in pediatric renal transplant recipients. Pediatr Nephrol. 2012;27(5):705-717.
   CrossRef - PubMed
   
4. Mueller K, Schachtner T, Sattler A, et al. BK-VP3 as a new target of cellular immunity in BK virus infection. Transplantation. 2011;91(1):100-107.
   CrossRef - PubMed
   
5. Womer KL, Huang Y, Herren H, et al. Dendritic cell deficiency associated with development of BK viremia and nephropathy in renal transplant recipients. Transplantation. 2010;89(1):115-123.
   CrossRef - PubMed
   
6. Comoli P, Basso S, Azzi A, et al. Dendritic cells pulsed with polyomavirus BK antigen induce ex vivo polyoma BK virus-specific cytotoxic T-cell lines in seropositive healthy individuals and renal transplant recipients. J Am Soc Nephrol. 2003;14(12):3197-3204.
   CrossRef - PubMed
   
7. Kariminik A, Yaghobi R, Dabiri S. Innate Immunity and BK Virus: Prospective Strategies. Viral Immunol. 2016;29(2):74-82.
   CrossRef - PubMed
   
8. Shabani Z, Bagheri M, Zare-Bidaki M, et al. NK cells in hepatitis B virus infection: a potent target for immunotherapy. Arch Virol. 2014;159(7):1555-1565.
   CrossRef - PubMed
   
9. Bauman Y, Nachmani D, Vitenshtein A, et al. An identical miRNA of the human JC and BK polyoma viruses targets the stress-induced ligand ULBP3 to escape immune elimination. Cell Host Microbe. 2011;9(2):93-102.
   CrossRef - PubMed
   
10. Trydzenskaya H, Juerchott K, Lachmann N, et al. The genetic predisposition of natural killer cell to BK virus-associated nephropathy in renal transplant patients. Kidney Int. 2013;84(2):359-365.
    CrossRef - PubMed
    
11. Fidan I, Rota S, Yesilyurt E, et al. Urinary cytokine response in BK virus infection. Gazi Med J. 2015;26:1-3.
    CrossRef
    
12. Gerdemann U, Christin AS, Vera JF, et al. Nucleofection of DCs to generate Multivirus-specific T cells for prevention or treatment of viral infections in the immunocompromised host. Mol Ther. 2009;17(9):1616-1625.
    CrossRef - PubMed
    
13. Womer KL, Peng R, Patton PR, et al. The effects of renal transplantation on peripheral blood dendritic cells. Clin Transplant. 2005;19(5):659-667.
    CrossRef - PubMed
    
14. Sadeghi M, Daniel V, Schnitzler P, et al. Urinary proinflammatory cytokine response in renal transplant recipients with polyomavirus BK viruria. Transplantation. 2009;88(9):1109-1116.
    CrossRef - PubMed
    
15. Geetha D, Levine SM, Manno RL, Valsamakis A, Ghazarian S, Seo P. BK virus replication in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Am J Nephrol. 2014;39(1):20-26.
    CrossRef - PubMed
    
16. Yamanaka K, Oka K, Nakazawa S, et al. Immunohistochemical features of BK virus nephropathy in renal transplant recipients. Clin Transplant. 2012;26 Suppl 24:20-24.
    CrossRef - PubMed
    
17. Delibrias CC, Kazatchkine MD, Fischer E. Evidence for the role of CR1 (CD35), in addition to CR2 (CD21), in facilitating infection of human T cells with opsonized HIV. Scand J Immunol. 1993;38(2):183-189.
    CrossRef - PubMed
    
18. Stimler NP, Hugli TE, Bloor CM. Pulmonary injury induced by C3a and C5a anaphylatoxins. Am J Pathol. 1980;100(2):327-348.
    PubMed
    
19. Fujita T. Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol. 2002;2(5):346-353.
    CrossRef - PubMed
    
20. Meymandi S, Dabiri S, Shamsi-Meymandi M, Nikpour H, Kharazmi A. Immunophenotypic pattern and cytokine profiles of dry type cutaneous leishmaniasis. Arch Iran Med. 2009;12(4):371-376.
    PubMed
    
21. Corallini A, Possati L, Trabanelli C, et al. Tumor-host interaction mediates the regression of BK virus-induced vascular tumors in mice: involvement of transforming growth factor-beta1. Carcinogenesis. 2003;24(9):1435-1444.
    CrossRef - PubMed
    
22. Abend JR, Low JA, Imperiale MJ. Inhibitory effect of gamma interferon on BK virus gene expression and replication. J Virol. 2007;81(1):272-279.
    CrossRef - PubMed
    
23. Wang H, Grzywacz B, Sukovich D, et al. The unexpected effect of cyclosporin A on CD56+CD16- and CD56+CD16+ natural killer cell subpopulations. Blood. 2007;110(5):1530-1539.
    CrossRef - PubMed
    
24. Lamarche C, Orio J, Collette S, et al. BK Polyomavirus and the Transplanted Kidney: Immunopathology and Therapeutic Approaches. Transplantation. 2016;100(11):2276-2287.
    CrossRef - PubMed
    
25. Hackstein H, Renner FC, Bohnert A, et al. Dendritic cell deficiency in the blood of kidney transplant patients on long-term immunosuppression: results of a prospective matched-cohort study. Am J Transplant. 2005;5(12):2945-2953.
    CrossRef - PubMed
    
26. Hesselink DA, Vaessen LM, Hop WC, et al. The effects of renal transplantation on circulating dendritic cells. Clin Exp Immunol. 2005;140(2):384-393.
    CrossRef - PubMed
    
27. Hirsch HH. BK virus: opportunity makes a pathogen. Clin Infect Dis. 2005;41(3):354-360.
    CrossRef - PubMed
    
28. Schachtner T, Muller K, Stein M, et al. BK virus-specific immunity kinetics: a predictor of recovery from polyomavirus BK-associated nephropathy. Am J Transplant. 2011;11(11):2443-2452.
    CrossRef - PubMed
    
29. Lukacher AE, Wilson CS. Resistance to polyoma virus-induced tumors correlates with CTL recognition of an immunodominant H-2Dk-restricted epitope in the middle T protein. J Immunol. 1998;160(4):1724-1734.
    PubMed
    
30. Comoli P, Hirsch HH, Ginevri F. Cellular immune responses to BK virus. Curr Opin Organ Transplant. 2008;13(6):569-574.
    CrossRef - PubMed
    
31. Comoli P, Basso S, Hirsch HH. Humoral and cellular immunity to polyomavirus BK large T and VP1 antigens after pediatric kidney transplantation. Am J Transplant. 2008;8(suppl 2):283.
    
    
32. Brennan DC, Agha I, Bohl DL, et al. Incidence of BK with tacrolimus versus cyclospoine and impact of preemptive immunosuppression reduction. Am J Transplant. 2005;5(3):582-594.
    CrossRef - PubMed
    
33. Binggeli S, Egli A, Schaub S, et al. Polyomavirus BK-specific cellular immune response to VP1 and large T-antigen in kidney transplant recipients. Am J Transplant. 2007;7(5):1131-1139.
    CrossRef - PubMed
    
34. Zhou W, Sharma M, Martinez J, et al. Functional characterization of BK virus-specific CD4+ T cells with cytotoxic potential in seropositive adults. Viral Immunol. 2007;20(3):379-388.
    CrossRef - PubMed
    
35. Harari A, Vallelian F, Meylan PR, Pantaleo G. Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence. J Immunol. 2005;174(2):1037-1045.
    CrossRef - PubMed
    
36. Chen Y, Trofe J, Gordon J, Autissier P, Woodle ES, Koralnik IJ. BKV and JCV large T antigen-specific CD8+ T cell response in HLA A*0201+ kidney transplant recipients with polyomavirus nephropathy and patients with progressive multifocal leukoencephalopathy. J Clin Virol. 2008;42(2):198-202.
    CrossRef - PubMed
    
37. Bodaghi S, Comoli P, Bosch R, et al. Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients. J Clin Microbiol. 2009;47(8):2577-2585.
    CrossRef - PubMed
    
38. Comoli P, Cioni M, Basso S, et al. Immunity to polyomavirus BK infection: immune monitoring to regulate the balance between risk of BKV nephropathy and induction of alloimmunity. Clin Dev Immunol. 2013;2013:256923.
    CrossRef - PubMed
    
39. Randhawa PS, Popescu I, Macedo C, et al. Detection of CD8+ T cells sensitized to BK virus large T antigen in healthy volunteers and kidney transplant recipients. Hum Immunol. 2006;67(4-5):298-302.
    CrossRef - PubMed
    
40. Kanto T, Inoue M, Miyatake H, et al. Reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize T helper cells in chronic hepatitis C virus infection. J Infect Dis. 2004;190(11):1919-1926.
    CrossRef - PubMed
    
41. Mengel M, Marwedel M, Radermacher J, et al. Incidence of polyomavirus-nephropathy in renal allografts: influence of modern immunosuppressive drugs. Nephrol Dial Transplant. 2003;18(6):1190-1196.
    CrossRef - PubMed
    
42. Han Lee ED, Kemball CC, Wang J, et al. A mouse model for polyomavirus-associated nephropathy of kidney transplants. Am J Transplant. 2006;6(5 Pt 1):913-922.
    CrossRef - PubMed
    
43. Cosio FG, Amer H, Grande JP, Larson TS, Stegall MD, Griffin MD. Comparison of low versus high tacrolimus levels in kidney transplantation: assessment of efficacy by protocol biopsies. Transplantation. 2007;83(4):411-416.
    CrossRef - PubMed
    
44. Araki K, Turner AP, Shaffer VO, et al. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009;460(7251):108-112.
    CrossRef - PubMed
    
45. Egli A, Kohli S, Dickenmann M, Hirsch HH. Inhibition of polyomavirus BK-specific T-Cell responses by immunosuppressive drugs. Transplantation. 2009;88(10):1161-1168.
    CrossRef - PubMed
    
46. Weist BJ, Wehler P, El Ahmad L, et al. A revised strategy for monitoring BKV-specific cellular immunity in kidney transplant patients. Kidney Int. 2015;88(6):1293-1303.
    CrossRef - PubMed
    
47. Pham PT, Schaenman J, Pham PC. BK virus infection following kidney transplantation: an overview of risk factors, screening strategies, and therapeutic interventions. Curr Opin Organ Transplant. 2014;19(4):401-412.
    CrossRef - PubMed
    
48. Costa C, Saldan A, Cavallo R. Evaluation of virus-specific cellular immune response in transplant patients. World J Virol. 2012;1(6):150-153.
    CrossRef - PubMed
    
49. Hammer MH, Brestrich G, Andree H, et al. HLA type-independent method to monitor polyoma BK virus-specific CD4 and CD8 T-cell immunity. Am J Transplant. 2006;6(3):625-631.
    CrossRef - PubMed
    
50. Blyth E, Clancy L, Simms R, et al. BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses. Transplantation. 2011;92(10):1077-1084.
    CrossRef - PubMed
    
51. Davies SI, Muranski P. T cell therapies for human polyomavirus diseases. Cytotherapy. 2017;19(11):1302-1316.
    CrossRef - PubMed
    
52. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science. 1992;257(5067):238-241.
    CrossRef - PubMed
    
53. Bollard CM, Heslop HE. T cells for viral infections after allogeneic hematopoietic stem cell transplant. Blood. 2016;127(26):3331-3340.
    CrossRef - PubMed
    
54. O'Reilly RJ, Prockop S, Hasan AN, Koehne G, Doubrovina E. Virus-specific T-cell banks for 'off the shelf' adoptive therapy of refractory infections. Bone Marrow Transplant. 2016;51(9):1163-1172.
    CrossRef - PubMed
    
55. Tzannou I, Papadopoulou A, Naik S, et al. Off-the-shelf virus-specific T cells to treat BK virus, human herpesvirus 6, cytomegalovirus, Epstein-Barr virus, and adenovirus infections after allogeneic hematopoietic stem-cell transplantation. J Clin Oncol. 2017;35(31):3547-3557.
    CrossRef - PubMed
    
56. Barrett AJ, Prockop S, Bollard CM. Virus-specific t cells: broadening applicability. Biol Blood Marrow Transplant. 2018;24(1):13-18.
    CrossRef - PubMed
    
57. Papadopoulou A, Gerdemann U, Katari UL, et al. Activity of broad-spectrum T cells as treatment for AdV, EBV, CMV, BKV, and HHV6 infections after HSCT. Sci Transl Med. 2014;6(242):242ra283.
    CrossRef - PubMed
    
58. Comoli P, Maccario R, Locatelli F, et al. Treatment of EBV-related post-renal transplant lymphoproliferative disease with a tailored regimen including EBV-specific T cells. Am J Transplant. 2005;5(6):1415-1422.
    CrossRef - PubMed
    
59. Schmitt A, Tonn T, Busch DH, et al. Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation. Transfusion. 2011;51(3):591-599.
    CrossRef - PubMed
    
60. Feuchtinger T, Opherk K, Bethge WA, et al. Adoptive transfer of pp65-specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation. Blood. 2010;116(20):4360-4367.
    CrossRef - PubMed
    
61. Khanna R, Smith C. Cellular immune therapy for viral infections in transplant patients. Indian J Med Res. 2013;138(5):796-807.
    PubMed
    
62. Ambalathingal GR, Francis RS, Smyth MJ, Smith C, Khanna R. BK Polyomavirus: Clinical Aspects, Immune Regulation, and Emerging Therapies. Clin Microbiol Rev. 2017;30(2):503-528.
    CrossRef - PubMed
    
63. Gerdemann U, Keirnan JM, Katari UL, et al. Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections. Mol Ther. 2012;20(8):1622-1632.
    CrossRef - PubMed
    
64. Dasari V, Schuessler A, Smith C, et al. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients. Mol Ther Methods Clin Dev. 2016;3:16058.
    CrossRef - PubMed
    
65. Mani J, Jin N, Schmitt M. Cellular immunotherapy for patients with reactivation of JC and BK polyomaviruses after transplantation. Cytotherapy. 2014;16(10):1325-1335.
    CrossRef - PubMed
    
66. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant. 2009;9 Suppl 3:S1-155.
    CrossRef - PubMed
    
67. Ramaswami B, Popescu I, Macedo C, et al. HLA-A01-, -A03-, and -A024-binding nanomeric epitopes in polyomavirus BK large T antigen. Hum Immunol. 2009;70(9):722-728.
    CrossRef - PubMed
    
68. Delbue S, Ferraresso M, Ghio L, et al. A review on JC virus infection in kidney transplant recipients. Clin Dev Immunol. 2013;2013:926391.
    CrossRef - PubMed
    
69. Balduzzi A, Lucchini G, Hirsch HH, et al. Polyomavirus JC-targeted T-cell therapy for progressive multiple leukoencephalopathy in a hematopoietic cell transplantation recipient. Bone Marrow Transplant. 2011;46(7):987-992.
    CrossRef - PubMed
    
70. Caparros-Wanderley W, Clark B, Griffin BE. Effect of dose and long-term storage on the immunogenicity of murine polyomavirus VP1 virus-like particles. Vaccine. 2004;22(3-4):352-361.
    CrossRef - PubMed
    
71. Husseiny MI, Lacey SF. Development of infectious recombinant BK virus. Virus Res. 2011;161(2):150-161.
    CrossRef - PubMed
    
72. Randhawa P, Viscidi R, Carter JJ, et al. Identification of species-specific and cross-reactive epitopes in human polyomavirus capsids using monoclonal antibodies. J Gen Virol. 2009;90(Pt 3):634-639.
    CrossRef - PubMed
    
73. Lakhashe SK, Thakar MR, Bharucha KE, Paranjape RS. Quantitation of HLA proteins incorporated by human immunodeficiency virus type 1 and assessment of neutralizing activity of anti-HLA antibodies. J Virol. 2008;82(1):428-434.
    CrossRef - PubMed
    
74. Maggiore U, Medici MC, Vaglio A, Buzio C. Increased viral load after intravenous immunoglobulin therapy for BK virus-associated nephropathy. Transpl Infect Dis. 2010;12(5):470-472.
    CrossRef - PubMed
    
75. Sharma AP, Moussa M, Casier S, Rehman F, Filler G, Grimmer J. Intravenous immunoglobulin as rescue therapy for BK virus nephropathy. Pediatr Transplant. 2009;13(1):123-129.
    CrossRef - PubMed