This single arm RZV immunogenicity study was conducted as part of a tertiary care lung transplant program at the University Health Network Transplant Centre, Toronto. Consecutive single or double lung transplant recipients who were ≥50 years of age were approached in outpatient transplant clinic from March 2018 to August 2018. Patients were eligible if they were ≥90 days posttransplant and had positive varicella zoster IgG antibodies before transplantation (see details in Supporting Information). The study was registered at ClinicalTrials.gov (NCT03493776).

After obtaining written informed consent, lung transplant recipients received two intramuscular doses of RZV (0.5 mL) 2–6 months apart. Serum and whole blood were obtained immediately before the first vaccination (baseline), immediately before the second vaccination (T1), and 3–6 weeks after the second vaccination (T2). The primary outcomes of the study were cellular and humoral immune responses to RZV vaccination. The secondary outcome was the safety of the vaccine.

The humoral immune response was evaluated using an enzyme-linked immunosorbent assay (ELISA) targeting VZV glycoprotein E (gE) antibodies at T1 and T2 as compared to baseline.¹³ Testing was performed at the VZV reference laboratory at the Centers for Disease Control and Prevention (CDC). Results were expressed as optical density (OD) values. Avidity testing of anti-gE antibodies was also performed.

In order to assess the cell-mediated immune response, we modified previously described methods.^14,15 The frequencies of gE-specific CD4²⁺ T cells (CD4+T cells expressing two or more activation markers of the four assessed: interferon-γ, interleukin-2, tumor necrosis factor–α, and CD40 ligand) were measured, after in vitro stimulation with a pool of one hundred and fifty-three 15-mer peptides (1.25 μg/mL each, overlapping by 11 amino acids) which span the entire VZV gE protein (JPT Peptide Technologies, Berlin, Germany), by intracellular cytokine staining and detection by flow cytometry as previously established by others.^12,16–22 Details are provided in the Supporting Information.

Standardized diaries were provided to all participants to record solicited local and general adverse events for 7 days after each vaccination (see details in Supporting Information). All solicited local and generalized symptoms were considered causally related to the vaccine. Unsolicited adverse events such as hospitalizations and rejection episodes were collected until 3 months after the last vaccine dose. For patients who had only one vaccine dose, unsolicited adverse events were collected until the latest possible time point for administration of the second dose (6 months after the first vaccine). Allograft rejection was defined as biopsy proven or clinically suspected and treated. We also performed a 2-year follow-up by chart review of these patients for the occurrence of shingles.

The sample size of 50 patients was based on feasibility and enrollment of at least 10% of the prevalent lung transplant population over age 50 years during the study period. Reactogenicity and safety results were analyzed in all participants who were vaccinated with at least one dose of RZV. The analyses of humoral and cellular immune responses were performed in patients for whom all baseline and postvaccine specimens were available (provided the PBMC counts allowed the assay to be performed) (Figure 1). For group comparisons, chi-squared or Fisher exact test was used for dichotomous variables, whereas Mann–Whitney U test was used for continuous variables. We used Wilcoxon signed rank test for paired analysis. Values of p < .05 were considered statistically significant. Statistics were done in GraphPad version 7.0 and in STATA version 16.0 (Stata Corp.). Graphs were made using GraphPad version 7.0 or National Institute of Health (NIH)’s Simplified presentation of incredibly complex evaluations (SPICE).²³

We enrolled a total of 50 lung transplant recipients. Of these, 49 patients received at least one vaccine dose (Figure 1). The median age of participants was 65.4 years (IQR: 59.1–70.6). Most participants were men (29/49; 59.2%) and most had a double vs. single lung transplant (40/49; 80.1%) (Table 1). The median time from transplant to the first vaccination was 3.0 years (IQR: 1.0–5.0). The median interval between the first and second vaccine was 63 days (IQR: 62–69). Except for one patient, all participants were on triple immunosuppressive treatment with median trough levels for cyclosporine (192 ng/mL; IQR: 155–227) or tacrolimus (12.5 ng/mL; IQR: 7.2–14.9) at time of the first vaccine.

Serum samples at all time points were available for 43 lung transplant recipients (Figure 1). The VZV gE antibody concentration increased significantly compared to baseline (median OD 1.96; IQR: 1.17–2.89) after the first (median OD 3.41, IQR 2.54–3.81, p < .0001) and the second vaccine dose (median OD 3.63, IQR 3.39–3.86, p < .0001; Figure 2A). At baseline, gE-specific antibody avidity was relatively low (46.4%, IQR: 36.6–63.4). Noteworthy, the avidity of the antibodies increased significantly after the first (71.5%; IQR: 52.9–91.5; p < .0001) and the second vaccine (84.2%; IQR: 59.4–96.5; p < .0001) (Figure 2B). There was a strong positive correlation between VZV anti-gE antibody concentrations (as by OD) and the avidity of anti-gE antibodies at baseline (Spearman's rho = 0.6442; p < .0001) after the first (Spearman's rho = 0.8325; p < .0001) and after the second vaccine (Spearman's rho = 0.7338; p < .0001). Further analysis did not show a significant correlation between anti-gE antibody concentrations and patient age or time from transplant to vaccination (data not shown) but showed an inverse correlation between dose of mycophenolate and antibody concentration (Spearman's rho = −0.3447; p = .019).

Cell-mediated immune responses were analyzed for 38 patients (Figure 1). VZV gE-specific CD4²⁺ T cell frequencies increased significantly from baseline (median 85 CD4²⁺ T cells per 10⁶ CD4T cells; IQR: 46–180) to after the first vaccine (median 128 CD4²⁺ T cells per 10⁶ CD4T cells; IQR 82–353; p = .023) and after the second vaccine (median 361 CD4²⁺ T cells per 10⁶ CD4T cells; IQR: 146–848; p < .0001) (Figure 3). Among VZV gE-specific CD4²⁺ T cells, IL-2+CD40L+ CD4 T cells contributed the most to the polyfunctional signature (Figure 4). There was no significant increase in VZV-specific CD8 T cells due to vaccination (data not shown). Patients with a longer interval between transplantation and vaccination had higher frequencies of CD4²⁺ T cells at baseline (Spearman's rho = 0.4118; p = .0102) and a trend toward higher gE-specific CD4²⁺ T cells after the second vaccine dose (Spearman's rho = 0.3184; p = .0514). There was no correlation with age and the frequencies of CD4²⁺ T cells at baseline or after vaccination (data not shown).

Anti-gE antibody concentrations (as by OD) and the number of gE-specific CD4²⁺ T cells were not correlated at any of the different time points (data not shown).

Forty-nine participants received at least one vaccine dose (included in the safety analysis for unsolicited adverse events). Of these, 47 of 49 completed the standardized diary for capturing solicited adverse events after the first vaccine dose and 44 of 45 completed the diary after the second vaccine dose.

During the 7-day postvaccination follow-up for solicited adverse events, the most common local adverse event was tenderness at injection site reported by 83.0% (95% CI: 69.2–92.4%) (39/47), followed by redness (31.9%; 95% CI: 19.1–47.1%) (15/47) and swelling (26.7%; 95% CI: 14.6–41.9%) (12/45). Only a minority of these events were classified as grade 3; all were self-limited (Figure 5). When occurring, local adverse events were of short duration. Tenderness at injection time resolved after a median of 2 days (IQR: 2–3 days), so did redness (IQR: 1–3 days) and swelling (IQR: 2–3 days). The most frequent generalized solicited symptoms were myalgia (36.2%; 95% CI 22.7–51.5; 17/47), fatigue (31.9%; 95% CI 19.1–47.1; 15/47), and headache (23.4%; 95% CI 12.3–38.0; 11/47). Gastrointestinal complaints (6.4%; 95% CI 1.3–17.5; 3/47) and shivering (4.3%; 95% CI 0.5–14.5; 2/47) were rare, and no participant developed fever within 7 days after injection.

From the first vaccination to months 3 after the last immunization, we recorded 26 unsolicited adverse events of which 14 were severe adverse events (Table E1 Supporting Information). Throughout the study, one death was reported (respiratory failure due to laboratory-confirmed influenza B infection) which we considered unrelated to vaccination. Two patients developed a HZ infection during the study 2-year follow-up for occurrence of shingles (with no cases occurring after the second vaccine dose): one participant experienced a mono-dermatomal infection 2 days after the first vaccine dose. The patient had treatment with valacyclovir and recovered without sequelae. In retrospect, this patient already had symptoms of reactivation (pain at reactivation site) at the time of vaccination. The second participant developed a disseminated herpes zoster episode 32 days after the first vaccine dose. This patient’s lesions resolved with intravenous acyclovir although he developed postherpetic neuralgia.

Three participants, all of whom were within the first year posttransplant, experienced four rejection episodes (none documented to be antibody-mediated). One lung transplant recipient had two episodes of clinically suspected rejection 89 and 130 days after the first vaccination (both episodes before the second vaccine dose), respectively. Due to the long interval between suspected rejection and RZV immunization, these rejection episodes were classified as unrelated to vaccination. Two participants experienced a histologically proven cellular rejection episode (both grade A1 according to the ISHLT classification²⁴) at 17 days and 90 days after second vaccination, respectively.

Protocol biopsies and DSA measurements for clinical purposes in our program were done at 1, 3, 6, 9, 12, 18, 24 months posttransplant and repeated if rejection was suspected. Based on this, there were 11 patients in the study cohort (all recently transplanted within the first 2 years) who had DSA measured in the 3 months prior to first vaccine and up to 3 months after the second dose of vaccine. Of these, only one patient developed new weak positive DSA compared to prevaccination. The patient was 9 months posttransplant and a biopsy done at that time showed no rejection.