ELISPOT assays with pp65 peptides or whole HCMV antigen are reliable predictors of immune control of HCMV infection in seropositive kidney transplant recipients

Abstract

Human cytomegalovirus (HCMV) infection represents a major complication for solid organ transplant recipients. The aim of this study was to verify if the measurement of HCMV‐specific T‐cells could help to identify patients protected against HCMV disease cytokine flow cytometry using infected dendritic cells as stimulus (CFC‐iDC, which discriminates between CD4⁺ and CD8⁺ T cells), and ELISPOT, using infected cell lysate (ELISPOT‐iCL) or pp65 (ELISPOT‐pp65) as stimulus, were adopted. Among the 47 kidney transplant recipients (KTR) enrolled, 29 had a self‐resolving HCMV infection (Controllers) and 18 required antiviral treatment (Non‐Controllers). HCMV‐specific T‐cell frequency at the peak of HCMV infection identified Controllers and Non‐Controllers, although the diagnostic performance of CD8⁺ CFC‐iDC (area under the curve [AUC] of the receiver‐operator characteristic curve: 0.65) was lower than that of CD4⁺ CFC‐iDC (AUC: 0.83), ELISPOT‐iCL (AUC: 0.83) and ELISPOT‐pp65 (AUC: 0.80). CFC‐iDC detected a protective immune reconstitution significantly earlier (median time: 38 days) than ELISPOT‐iCL and ELISPOT‐pp65 (median time: 126 and 133 days, respectively). Time to protective immune reconstitution in Non‐Controllers was significantly longer than in Controllers with the ELISPOT and the CD4⁺ CFC‐iDC assays, but not with CD8⁺ CFC‐iDC. The majority of patients did not require antiviral treatment after protective immune reconstitution, with the exception of five patients according to CFC‐iDC assay, one patient according to ELISPOT‐iCL assay and three patients according to ELISPOT‐pp65 assay. Monitoring the HCMV‐specific immunological reconstitution with is effective in discriminating KTR at risk of or protected from HCMV disease and the ELISPOT assays are suitable for implementation in the clinical setting.

1. INTRODUCTION

Human cytomegalovirus (HCMV) is a complex DNA virus that undergoes latency following the primary infection. Latent strain can periodically restart replication causing reactivation episodes. Additionally, a new HCMV strain can also superinfect an already immune individual by causing reinfection. HCMV infection represents a major complication for solid organ transplant recipients (SOTR) and hematopoietic stem cells (HSCTR) with serious life‐threatening risks. ¹ , ² Due to the immune suppression to which transplant recipients are subjected, these patients can reactivate the latent HCMV (either from their own organism or from the transplanted organ), and the infection can result in disseminated disease (systemic syndrome) or tissue invasive disease (TID). Currently, two strategies are adopted to prevent HCMV disease: the universal prophylaxis (i.e., administration of antiviral drugs to all transplanted patients for 6–12 months) and the active surveillance with pre‐emptive therapy administration (i.e., monitoring the blood viral load and giving antiviral drugs to patients at predetermined levels of viral load). However, several patients may avoid both prophylaxis and active surveillance through virological monitoring, because they do not undergo significant HCMV‐specific T‐cell impairment or have early reconstitution of an efficient immune system capable of controlling HCMV reactivation. ³ , ⁴ , ⁵ The relative contribution of the humoral and T‐cell immunity in limiting the spread of HCMV is still unknown and the possible protection provided by antibodies is debated. ⁶ , ⁷ , ⁸ It has been observed that HCMV TID occurs rarely in patients who reconstituted HCMV‐specific T‐cells compartment in peripheral blood, although TID may occur also in the presence of apparently efficient virus‐specific T‐cells in peripheral blood and in the absence of detectable viremia. ⁹ , ¹⁰

The aim of this study was to verify if the measurement of HCMV‐specific T‐cells can help to identify patients protected against HCMV disease and distinguish them from those who are at risk. In this case, it would be possible to defer therapeutic intervention or virological monitoring in patients with an efficient HCMV‐specific immunological response. Three different assays were evaluated and compared with this purpose. Cytokine flow cytometry using infected dendritic cells as stimulus (CFC‐iDC) and ELISPOT, using infected cell lysate (ELISPOT‐iCL) were already analyzed for their performance in predicting immune protection against HCMV infection in previous cohorts of SOTR, while the ELISPOT, using pp65 (ELISPOT‐pp65) assay was analyzed in this study.

2. METHODS

2.1. Patients enrolled and management of HCMV infection

The study was designed to verify if the measurement of HCMV‐specific T‐cell frequency with three different assays can help to identify patients protected or at risk of developing HCMV disease. The primary objective was the number of patients requiring antiviral treatment after the detection of HCMV‐specific T‐cell frequency above the cutoff indicating protective immunity according to each assay (i.e., failure of the immunological assay).

Between June 2018 and December 2019, 47 HCMV seropositive KTR (kidney transplant recipients) were enrolled in the study at Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. Patients were monitored for HCMV DNAemia in whole blood, ¹¹ weekly for the first 8 weeks and subsequently every 15 days until the 4th month, then monthly until 1 year posttransplantation.

In case of suspected TID, a tissue biopsy (gastrointestinal disease) or a bronco‐alveolar lavage (BAL) fluid sample (pneumonia) was collected for HCMV DNA quantification and histopathological analysis. HCMV disease was defined as possible, probable, or proven according to Ljungman et al. ¹²

Antiviral treatment with ganciclovir (GCV) or valganciclovir (VGCV) was administered pre‐emptively, after the detection of 300 000 HCMV DNA copies/ml whole blood, or in case of suspected or diagnosed HCMV disease.

Total and HCMV‐specific T‐cell immunity (CD4⁺ and CD8⁺) was determined once per month until 4 months and then every 2 months until 1 year posttransplantation.

2.2. CFC‐iDC assay

Each patient was tested at all time points by CFC‐iDC. ¹⁰ , ¹³ , ¹⁴ Our in‐house developed CFC‐iDC assay was based on 24 h coculture of patients' autologous immature DC, which were infected with endotheliotropic and leukotropic HCMV strain VR1814, with peripheral blood mononuclear cells. Then, both activated CD4⁺ and CD8⁺ T‐cells were quantified by cytokine flow cytometry analysis of intracellular IFN‐γ production. ¹⁰ , ¹³ The frequency of IFN‐γ‐producing CD4⁺ and CD8⁺ T‐cells is determined by subtracting the frequency of PBMCs incubated with no infected DCs (<0.05%) from the frequency of PBMCs incubated with HCMV‐infected DCs. ¹³ The total number of HCMV‐specific CD4⁺ and CD8⁺ T‐cells were calculated by multiplying the percentage of HCMV‐specific T‐cells positive for IFN‐γ production by the corresponding absolute CD4⁺ and CD8⁺ T‐cell count. Levels of HCMV‐specific CD4⁺ and CD8⁺ T‐cells above 0.4 cells/μL and 2 cells/μL of blood are considered protective. This cut‐off is based on the analysis of results from 199 patients enrolled in two previous studies. ⁴ , ¹⁵

2.3. ELISPOT‐iCL assay

A 24‐h ELISPOT assay was performed with a commercial CE‐marked kit for IFN‐γ detection (Autoimmune Diagnostika), modified using as a stimulus a commercially available HCMV AD169 infected cell lysate (Microbix Biosystem) as described previously. ¹³ ELISPOT images were acquired and analyzed using an automated image scanner (iSpot Reader Spectrum, AID) The total number of HCMV‐specific spot‐forming T‐cells/µL was calculated with the formula (net spot number x  lymphocyte number/µL blood)/2 × 10⁵. ¹⁶ Subjects with at least 0.1 HMCV‐specific T‐cells/µL were considered “protected”; this cut‐off was previously validated. ¹³

2.4. ELISPOT‐pp65 assay

Human IFN‐γ ELISPOT kits (Diaclone) and Multiscreen‐IP membrane‐bottomed 96‐well plates (Merck Millipore) were used as described. ¹⁶ , ¹⁷ Briefly, plates were coated overnight with monoclonal capture antibody against IFN‐γ and stored at 4°C. After washing with PBS, plates were blocked with culture medium for 2 h at room temperature. Cells were plated in duplicate (1 × 10⁵/100 μL per well) and stimulated with pp65 peptides pool (JPT Peptide Technologies) at final concentration (0.25 µg/mL) or with phytohemagglutinin (PHA; Sigma‐Aldrich) at final concentration (5 µg/mL) or with medium alone (negative control) and incubated at 37°C in a 5% CO₂ humidified atmosphere for 24 h. After washing, plates were incubated overnight at 4°C with biotinylated IFN‐γ detection antibody. Plates were washed, streptavidin‐alkaline phosphatase conjugate was added, and plates were incubated at 37°C in a 5% CO₂ atmosphere for 1 h. Plates were then washed, and 5‐bromo‐4‐chloro‐3‐indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) was added for 20 min at room temperature. Wells were then washed several times under running water and air‐dried overnight. Spots were counted by using an automated AID ELISPOT reader system (Autoimmun Diagnostika GmbH). The mean number of spots from duplicate wells was adjusted to 1 × 10⁶ PBMCs. The net spots per million PBMCs was calculated by subtracting the number of spots responding to the negative control from the number of spots responding to the corresponding antigen and results were given as net spots/million PBMCs. The total number of HCMV‐specific spot‐forming T‐cells/µL was calculated with the formula (net spot number × lymphocyte number/µL blood)/2 × 10⁵. The protective cut‐off was calculated on data obtained concomitantly to HCMV DNA peak level in patients controlling spontaneously HCMV infection or requiring antiviral treatment (see Section 3).

2.5. Statistical analysis

Receiver‐operator characteristic (ROC) analysis was used to assess the performance of the three immunological assays in predicting the spontaneous control of HCMV infection. The area under the curve (AUC) and its 95% confidence interval (CI) were calculated. The cut‐off indicating protection from HCMV infection for ELISPOT‐pp65 was established according to the Concordance Probability Method; therefore, the cut‐off expressing the highest values of sensitivity multiplied by specificity was selected. The cumulative incidence of HCMV‐specific T‐cell immunity reconstitution (i.e., development of HCMV‐specific T‐cell frequency above the protective cut‐off) according to the three assays was determined using Kaplan–Meier curves, which were compared by the log‐rank (Mantel‐Cox) test. Student's t test was used to compare HCMV DNA viral load and T‐cell frequency, in the two groups of patients, at different time points, after the data were log‐transformed.

3. RESULTS

3.1. Patients' characteristics

Of the 47 patients enrolled, 43 patients were followed until 1 year after transplantation, while 4 patients concluded the study earlier (243–297 days after transplant) due to death for fungal pneumonia (n = 1, see below) or because the patients were transferred to other centers (n = 3). Twenty‐nine (62%) patients had self‐resolving HCMV infection and were named Controllers, while 18 (38%) patients were treated for systemic infection and/or TID with antiviral drugs and were named Non‐Controllers.

Patients' characteristics are shown in Table 1. There were no significant differences in baseline characteristics among the two groups of Controllers and Non‐Controllers, except for the older age of Non‐Controllers (p = 0.05) (Table 1).

Table 1.

Patients' characteristics.

Characteristics	All patients (n = 47)	Controllers (n = 29)	Non‐Controllers (n = 18)	p
Age, median (range)	49 (25–76)	46 (25–67)	57 (38–76)	0.05
Gender, n (%)
Male	31 (66)	18 (62)	13 (72)	0.80
Female	16 (34)	11 (38)	5 (28)
Nephropathy, n (%)
Polycystic kidney	8 (18)	3 (10)	5 (28)	0.22
Glomerulonephritis	10 (22)	9 (31)	1 (6)	0.06
Nephropathy	7 (15)	6 (21)	1 (6)	0.22
Nephroangiosclerosis	4 (6)	3 (10)	1 (6)	1.00
Diabetes	2 (4)	0	2 (11)	0.14
Other	6 (13)	4 (14)	2 (11)	1.00
Unknown	10 (22)	4 (14)	6 (32)	0.15
Induction therapy, n (%)
Basiliximab	40 (85)	23 (79)	17 (94)	0.22
ATG	7 (15)	6 (21)	1 (6)
Maintenance therapy, n (%)
FK506	41 (87)	26 (90)	15 (83)	0.66
Cyclosporine A	5 (11)	2 (7)	3 (17)	0.35
MMF	45 (96)	27 (93)	18 (100)	0.51
MPRE	47 (100)	29 (100)	18 (100)	1.00
Everolimus	1 (2)	1 (3)	0	1.00

Abbreviations: ATG, antihuman thymocyte globulin; FK506 tacrolimus; MMF, mycophenolate mofetil; MPRE, methylprednisolone.

The level of HCMV DNA peak, and the time to reach the peak in the 29 Controllers and in the 18 Non‐Controllers is shown in Figure 1A,B. Controllers showed the HCMV DNA peak after a median time of 80 days, while Non‐Controllers showed the HCMV DNA peak after a median time of 52 days (p = 0.457; Figure 1B).

Figure 1.

Human cytomegalovirus (HCMV) DNA peak value and timing. (A) HCMV DNA blood peak in Controllers (solid green circle) and Non‐Controllers (solid red or yellow circle). Patients treated for tissue‐invasive disease are indicated by solid yellow circle. (B) Time to HCMV DNA peak in Controllers (solid green line) and Non‐Controllers (solid red line) (median time 80 and 52 days, respectively).

3.2. Identification of Controllers and Non‐Controllers by the three assays and calculation of the cut‐off for HCMV immune protection with the ELISPOT‐pp65 assay

Using a ROC curve analysis, we compared the diagnostic performance of three different immunological assays (CFC‐iDC, ELISPOT‐iCL, and ELISPOT‐pp65) in the identification of Controllers and Non‐Controllers at the time of HCMV DNA peak (Figure 2).

Figure 2.

Identification Controller (solid green circle) and Non‐Controllers (solid red circle) by three assays and calculation immune cut‐off. (A) Protective immune cut‐off with CD4⁺ CFC‐iDC assay. (B) CD4⁺ CFC‐iDC ROC curve. (C) Protective immune cut‐off with CD8⁺ CFC‐iDC assay. (D) CD8⁺ CFC‐iDC ROC curve (E) Protective immune cut‐off with ELISPOT‐iCL assay. (F) ELISPOT‐iCL ROC curve. (G) Protective immune cut‐off with ELISPOT‐pp65 assay. (H) ELISPOT‐pp65 ROC curve. CFC‐iDC, cytokine flow cytometry using infected dendritic cells as stimulus; ELISPOT‐iCL; ELISPOT, using infected cell lysate; ROC, receiver‐operator characteristic; ELISPOT‐pp65, ELISPOT, using pp65.

CD4⁺ CFC‐iDC assay and ELISPOT‐iCL showed the best values of the AUC, that is, 0.83 (95% CI: 0.71–0.95) for both assays (Figure 2A,B,E,F), while the AUC for ELISPOT‐pp65 was slightly inferior (0.80; 95% CI: 0.66–0.94) (Figure 2G,H). Instead, the AUC for CD8⁺ CFC‐iDC was the worst (0.65; 95% CI: 0.48–0.83) (Figure 2C,D).

For the ELISPOT‐pp65 assay, the cut‐off 0.5 pp65‐specific T‐cells/μL was selected as the one showing the maximal level of sensitivity and specificity in the identification of Controllers, whereas the protective cut‐offs 0.4 T‐cells/μL for HCMV‐specific CD4⁺ and 2 T‐cell/μL for HCMV‐specific CD8⁺ T‐cells for the CFC‐iDC assay and 0.1 T‐cells/μL for ELISPOT‐iCL assay, ¹³ were determined previously.

3.3. Kinetics of HCMV DNA in blood and T‐cell immunity reconstitution with CFC‐iDC, ELISPOT‐iCL, and ELISPOT‐pp65 assays

The average kinetics of HCMV DNAemia and HCMV‐specific T‐cells during the entire 1‐year follow‐up in the two groups of KTR is reported in Figure 3. Interestingly, when DNAemia levels (Figure 3A) were compared with HCMV‐specific CD4⁺ T‐cell levels at different time points, it was observed that in Controllers (green broken line), the presence of a consistent CD4⁺ T‐cell frequency since the beginning of the posttransplant period (Figure 3B) determined DNAemia disappearance at 180 days (Figure 3A), while in Non‐Controllers (red continue line), a significant drop in occurred in concomitance with HCMV DNA peak (60 days); subsequently, the CD4⁺ T‐cell reconstitution occurred at 180–360 days after transplant (Figure 3B), thus causing DNAemia disappearance at 360‐day time‐point (Figure 3A). It is worthy to note that, unlikely HCMV‐specific CD4⁺ T‐cell kinetics, specific CD8⁺ T‐cell immunity did not show a major drop in concomitance to HCMV DNA peak also in Non‐Controllers (Figure 3B). For the T‐cell frequency determined with ELISPOT‐iCL and ELISPOT‐pp65, in Controllers the presence of specific T‐cells was sustained for the entire posttransplant period (Figure 3D,E), while in Non‐Controllers the drop of the T‐cell response and subsequent reconstitution were associated with the peak of HCMV DNA at 60‐day time point and its disappearance at 360‐day time point. The comparison of the Kaplan–Meier curves relevant to the recovery of T‐cell immunity according to the protective cut‐offs of each assay (see above) shows that the reconstitution process occurs with significantly different rates with the three different assays (Figure 4A). The CFC‐iDC assay showed significantly earlier protective immune reconstitution than the ELISPOT‐iCL and ELISPOT‐pp65 assays (p < 0.003; Figure 4A): the CFC‐iDC assay showed protective immune reconstitution after a median time of 38 days, while the ELISPOT‐iCL and ELISPOT‐pp65 assays showed protective immune recovery after a median time of 126 and 133 days, respectively.

Figure 3.

Kinetics of HCMV DNAemia and antigen‐specific T‐cell response in Controllers (solid green circle) and Non‐Controllers (solid red circle) at different time points (pretransplantation, 30, 60, 90, 180, and 360 days) after transplantation. (A) Non‐Controllers showed a significantly higher levels of HCMV DNAemia than Controllers at any time point. (B) Non‐Controllers showed a significantly low number of CFC‐iDC CD4⁺ (at 60, 90, and 180 days) than Controllers. (C) Non‐Controllers showed a significantly low number of CFC‐iDC CD8⁺ (at 60 and 90 days) than Controllers. (D) Non‐Controllers showed significantly low levels of T‐cell response (60, 90, and 180 days) with ELISPOT‐iCL than Controllers. (E) Non‐Controllers showed significantly low levels of T‐cell response (60, 90, 180, and 360) with ELISPOT‐pp65 than Controllers. Circles indicate mean log₁₀ value of HCMV DNA or T‐cell frequency at each time point (±15 days); vertical lines indicated standard deviation. Horizontal dot lines indicate cut‐off value for protective T‐cell frequency. *p < 0.05; **p < 0.01; ***p < 0.001. CFC‐iDC, cytokine flow cytometry using infected dendritic cells as stimulus; ELISPOT‐iCL, ELISPOT, using infected cell lysate; ELISPOT‐pp65, ELISPOT, using pp65; HCMV, human cytomegalovirus.

Figure 4.

Time for recovery of HCMV‐specific immunity in kidney transplanted patients. (A) Immune recovery in patients by three tests: the CFC‐iDC (orange line) assay showed protective immune recovery after a median time of 38 days, while the ELISPOT‐iCL (blue line) and ELISPOT‐pp65 (purple line) assays showed protective immune recovery after a median time of 126 and 133 days, respectively. (

B) Median time to immune recovery with CFC‐iDC assay was 31 days in Controllers (green line) and 101 days in Non‐Controllers (red line). (C) Median time to immune recovery with ELISPOT‐iCL assay was 66 days in Controllers and 170 days in Non‐Controllers. (D) Median time to immune recovery with ELISPOT‐pp65 assay was 87 days in Controllers and 188 days in Non‐Controllers. CFC‐iDC, cytokine flow cytometry using infected dendritic cells as stimulus; ELISPOT‐iCL, ELISPOT, using infected cell lysate; ELISPOT‐pp65, ELISPOT, using pp65; HCMV, human cytomegalovirus.

The time required to reach the protective level of HCMV‐specific T‐cell immunity in Non‐Controllers was significantly longer compared to Controllers with all three assays (Figure 4B–D). In particular, with the CFC‐iDC assay, Controllers showed protective immune recovery after a median time of 31 days, while Non‐Controllers showed protective immune reconstitution after a median time of 101 days (p < 0.004; Figure 4B). Instead, the median time for reaching protective immune recovery with the ELISPOT‐iCL assay was 66 days for Controllers and 170 days for Non‐Controllers (p = 0.034; Figure 4C). Similarly, the median time for reaching protective immune reconstitution was 87 days for Controllers and 188 days for Non‐Controllers (p = 0.005) with the ELISPOT‐pp65 assay (Figure 4D).

All Controllers had protective immune reconstitution with the CFC‐iDC assay at the end of follow‐up, while one Non‐Controller did not recover protective immunity during the study period (374 days after transplant). Instead, with the ELISPOT‐iCL assay, two Controllers and five Non‐Controllers did not show protective immune reconstitution at the end of follow‐up, while two Controllers and nine Non‐Controllers did not show protective immune recovery with the ELISPOT‐pp65 assay at the end of follow‐up.

Regarding the HCMV‐specific T‐cell immunity studied with the CFC‐iDC assay, it was possible to differentiate CD4⁺ and CD8⁺ T‐cell responses: Controllers showed earlier CD4⁺ T‐cell recovery than Non‐Controllers, with a median time of 31 and 101 days, respectively (p = 0.004; Figure 5A). However, no differences were observed between the two groups for the HCMV‐specific CD8⁺ T‐cell immunity (Figure 5B).

Figure 5.

T‐cell reconstitution in Controllers and Non‐Controllers by CFC‐iDC assay. (A) Controllers showed earlier CD4⁺ T‐cell recovery than Non‐Controllers (median time: 31 and 101 days, respectively). (B) Controller and Non‐Controllers showed the same CD8⁺ T‐cell recovery (30 and 33 days, respectively). CFC‐iDC, cytokine flow cytometry using infected dendritic cells as stimulus.

3.4. Development of immune response and treatment of HCMV infection

Thirteen of the 18 Non‐Controllers (72.2%) required antiviral treatment when the specific protective HCMV immunity was not yet acquired (Table 2). Five patients (WP4‐017; WP4‐033; WP4‐059; WP4‐061; and WP4‐087) were treated after protective immune reconstitution according to the CFC‐iDC assay (two patients with TID), one patient (WP4‐017) was treated after protective immune reconstitution according to the ELISPOT‐iCL assay and three patients (WP4‐017; WP4‐033; and WP4‐065) were treated after protective immune recovery according to the ELISPOT‐pp65 assay (two patients with TID).

Table 2.

Clinical and immune‐virological characteristics of patients treated for systemic infection and/or tissue‐invasive disease (TID).

Patients	Immune reconstitution (days) according to:			Pre‐emptive therapy		HCMV tissue invasive disease
	CFC‐iDC	ELISPOT‐iCL	ELISPOT‐ pp65	Onset of therapy after transplantation (days and symptoms)	HCMV DNA levels in the blood (copies/mL)	Definition	Onset of therapy after transplantation (days and symptoms)	Istopathology report	IHC	HCMV DNA levels in the tissue biopsy (copies/105 cells) (tissue)	HCMV DNA in the blood (copies/mL)
WP4‐017	25a	25a	25a	153 (diarrhea)	5400
WP4‐033	29a	80	29a	No	No	Probable	51 (epigastric pain)	Foveolar hyperplasia, mild chronic inflammation, mild fibrosis	Negative	3018 (stomach)	126 000
WP4‐059	35a	70	188	49 (vomiting)	545 400	Probable	128 (vomiting)	Foveolar hyperplasia, mild sclerosis	nd	1187 (stomach)	3510
WP4‐061	29a	106	Not detected (last follow‐up: 187)	73 (asymptomatic)	334 800				na
WP4‐087	24a	165	300	67 (asymptomatic)	174 600				na
WP4‐065	78	78	30a	No	No	Probable	44 (abdominal pain)	Mild chronic inflammation, focal intraepithelial granulocytes	Negative	632 (intestine)	3060
WP4‐003	365	122	Not detected (last follow‐up: 158)	131 (asymptomatic)	489 600				na
WP4‐007	216	294	Not detected (last follow‐up: 237)	No	No	Proven	237 (diarrhea, dysuria, and stranguria [concomitant bacterial UTI]).	Foveolar hyperplasia, chronic interstitial inflammation with lymphoid microaggregates	Positive	61 571 (stomach)	53 280
WP4‐028	115	158	295	57 (asymptomatic)	255 600				nd
WP4‐045	87	241	140	53 (epigastric pain)	56 790				na
WP4‐050	132	175	175	53 (asymptomatic)	246 600	Probable	284 (interstitial pneumonia [concomitant fungal infection])		na	83 700 (BAL)	1260
WP4‐068	203	367	Not detected (last follow‐up: 104)	84 (asthenia and diarrhea)	50 670				na
WP4‐074	85	132	Not detected (last follow‐up: 175)	47 (diarrhea, abdominal pain)	604 800				na
WP4‐081	Not detected (last follow‐up: 374)	Not detected (last follow‐up: 374)	Not detected (last follow‐up: 187)	65 (diarrhea)	157 590				na
WP4‐083	121	Not detected (last follow‐up: 369)	113	57 (asymptomatic)	549 900				na
WP4‐086	365	Not detected (last follow‐up: 365)	Not detected (last follow‐up: 365)	No	No	Proven	59 (epigastric pain, asthenia)	Erosive gastritis, necrosis and active inflammation, foveolar hyperplasia, sclerosis	Positive	31 038 (stomach); 614 974 (duodenum)	734 400
WP4‐089	249	Not detected (last follow‐up: 365)	Not detected (last follow‐up: 120)	96 (asymptomatic)	206 100				na
WP4‐094	83	Not detected (last follow‐up: 368)	Not detected (last follow‐up: 368)	41 (epigastric pain)	692 100				na

Abbreviations: CFC‐iDC, cytometry using infected dendritic cells as stimulus; ELISPOT‐iCL, ELISPOT, using infected cell lysate; HCMV, human cytomegalovirus; ELISPOT‐pp65, ELISPOT, using pp65; IHC, immune histochemistry; na, not available; nd, not done; UTI, urinary tract infection.

^a Patients treated after immune recovery.

In detail, among the 18 Non‐Controllers (Table 2), 14 patients received pre‐emptive antiviral therapy at a median time of 57 (range 53–96) days after transplantation; 7 patients were asymptomatic, 7 patients showed nonspecific gastrointestinal symptoms (abdominal or epigastric pain, vomiting, diarrhea), although TID was not demonstrated. HCMV DNA was detected in tissue/BAL samples in seven patients, but only in two of them immune histochemistry revealed a positive staining for HCMV. It is interesting to note that TID was not always associated with high levels of viremia in blood.

4. DISCUSSION

Results of the present study show that three different assays for the determination of HCMV‐specific T‐cell response can be efficiently adopted to identify KTR with immune protection against HCMV disease despite immune suppressive therapy. The flow‐cytometry‐based assay using as stimulus HCMV‐infected DC (CFC‐iDC) appears more sensitive in detecting immune protection, whereas, on the other hand, the two ELISPOT assays appear slightly more specific. The two ELISPOT assays: ELISPOT‐iCL and ELISPOT‐pp65, provide overlapping results, despite the different stimuli adopted.

Immunological monitoring with the CFC‐iDC assay was very sensitive and able to detect the HCMV‐specific T‐cell recovery after transplantation earlier, but this technique requires a 7‐day turnaround time and skills in cell culture and live virus handling.

The earlier detection of immune recovery by the CFC‐iDC assay can be ascribed to the more comprehensive detection of the T‐cell response against the whole HCMV proteome that can be presented by the infected cells, instead of the analysis of T‐cells specific for selected antigens or infected cell lysate. ¹³ , ¹⁸ However, during the first 1–2 months after transplantation, patients may not actually be protected despite the in vitro detection of HCMV‐specific T‐cells, as already observed in previous studies by our group ⁴ , ¹⁵ Therefore, the higher sensitivity in detecting an early immune recovery may not be a true advantage for the identification of protected patients.

The actual advantage of this assay, and in general of the flow cytometry‐based assays (regardless of the stimulus adopted), is the ability to discriminate between CD4⁺ and CD8⁺ responses because the long‐term protection from HCMV infection is achieved when the CD4⁺ T‐cell response is restored. In fact, CD8⁺ T‐cells are not protective in the absence of the CD4⁺ T‐cell counterpart ¹⁹ , ²⁰ and other authors reported a correlation between low CD4⁺ specific T‐cell response and HCMV events. ²¹ , ²² Monitoring with ELISPOT techniques is a method that can be more easily introduced into routine diagnostics and is faster in providing a response to the clinician, although there is later identification of protected patients with respect to the iDC‐CFC assay. In our KTR cohort, patients developing HCMV TID or requiring antiviral therapy for pre‐emptive purposes were scored as “unprotected” according to the ELISPOT assays, except for three cases: two patients with a diagnosis of probable HCMV gastritis and one patient who received pre‐emptive therapy but in noncompliance with the established protocol due to the very low viral load.

The fact that immune‐suppressed patients may develop HCMV TID even in the presence of HCMV‐specific T‐cells in peripheral blood was already observed in SOTR and in HSCTR. ¹⁰ , ²³ , ²⁴ Regarding this issue, it is worth noting that HCMV TID may occur in the presence of either high or low (or even undetectable) viral load in blood, ⁹ and therefore, tissue biopsy examination is required whenever HCMV TID is suspected. ¹²

The ELISPOT assays with iCL or pp65‐derived peptides provided overlapping results. However, although, ELISPOT‐iCL appears slightly more discriminatory, ELISPOT‐pp65 assays are easier to standardize, because involve the use of synthetic peptides, and are commercially available. ²⁵ Therefore, these assays may be suitable for a broader usage in the clinical setting.

The management of CMV disease for the immunosuppressed patients is different in the various centers and is essentially based on prophylaxis or active surveillance and pre‐emptive therapy, which is less frequently applied. ²⁶ , ²⁷ , ²⁸ Until now, active surveillance is based on the monitoring of HCMV DNA in blood or plasma, weekly in the first 3 months after transplantation and less frequently thereafter, since the effect of antirejection immune suppressive therapy is declining. The future approach could be to customize not only the start of antiviral therapy but also the follow‐up schedule based on immunological reconstitution, by providing more frequent viro‐immunological checks in patients without protective immunity, and reducing the controls in protected patients. This clinical monitoring is in line with the much‐desired personalized precision medicine. ²⁹ Similarly, the duration of prophylaxis could be tailored on HCMV‐specific immune reconstitution. The advantage of guiding the preventive measures by immunological surveillance lies mainly in sparing the use of antiviral drugs and their relevant costs and toxicity, ³⁰ especially hematological toxicity, which in turn exposes the patient to risk of infection or rejection. ³¹ In the past, our group has already devised the possibility of a new antiviral surveillance modality, personalizing the pre‐emptive therapy approach on the basis of immunological reconstitution with CFC‐iDC, ⁴ , ³² and this strategy was attempted preliminarily in HSCTR. ³ More recent clinical trials endorsed the personalization of the duration of anti‐HCMV prophylaxis on HCMV‐specific immune reconstitution in lung recipients or KTR. ³³ , ³⁴ , ³⁵ These studies adopted the QuantiFERON‐CMV assay, which measures mainly HCMV‐specific CD8⁺ T‐cell response by an interferon‐gamma release assay based on whole blood stimulation with a pool of epitopic peptides from different HCMV antigens. However, the QuantiFERON‐CMV assay showed a lower specificity than both the CFC‐iDC and ELISPOT‐pp65 assays in identifying patients able to control HCMV infection spontaneously. ²⁴ , ²⁵

These preliminary data make it possible to assert that monitoring the HCMV‐specific immunological reconstitution with an ELISPOT assays is effective in discriminating KTR at risk of or protected from post‐transplant HCMV disease and the assay can be implemented in transplantation centers for the personalization of HCMV prevention strategies. Results of this study deserve to be validated in prospective clinical trials aimed at tailoring active surveillance or antiviral prophylaxis in a larger cohort of patients.

AUTHOR CONTRIBUTIONS

Daniele Lilleri designed the study; Federica Zavaglio, Irene Cassaniti, Elisa Gabanti, and Marilena Gregorini analyzed and interpreted the data, and drafted the manuscript; Marilena Gregorini, Francesca Rivela, Elisa Gabanti, and Anna L. Asti collected and managed the data; Federica Zavaglio and Francesca Arena performed the experiments on T‐cell response; Marilena Gregorini enrolled the participants; Teresa Rampino and Fausto Baldanti supervised the study. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS APPROVAL STATEMENT

The study was approved by the Ethics Committee and Fondazione IRCCS Policlinico San Matteo Institutional Review Board (Procedure no 201800034325) and patients gave written informed consent.

Zavaglio F, Rivela F, Cassaniti I, et al. ELISPOT assays with pp65 peptides or whole HCMV antigen are reliable predictors of immune control of HCMV infection in seropositive kidney transplant recipients. J Med Virol. 2023;95:e28507. 10.1002/jmv.28507

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding authors upon reasonable request.