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Tiza–Titre increase and enhanced immunity through an adjuvanted, recombinant herpes zoster subunit vaccine in patients with liver cirrhosis and post-liver transplantation: a study protocol for a prospective cohort study
  1. Stephanie Vollmer-Raschdorf1,
  2. Jassin Rashidi-Alavijeh2,
  3. Sebastian Voigt3,
  4. Hartmut Hengel4,5,
  5. Benjamin Borchardt1,
  6. Daniela Huzly4,5,
  7. Eva-Maria Hüßler6,
  8. Jürgen in der Schmitten1,
  9. Anne Halenius4,
  10. Katharina Willuweit2,
  11. Suzan Botzenhardt7,
  12. Mirko Trilling3,
  13. Tobias Boettler8,
  14. Dorothea Dehnen1
  1. 1Institute of General Practice, Medical Faculty, University of Duisburg-Essen, Essen, Germany
  2. 2Department of Gastroenterology and Hepatology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
  3. 3Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
  4. 4Institute of Virology, Freiburg University Medical Centre, Faculty of Medicine, University of Freiburg, Freiburg, Germany
  5. 5German Consulting Laboratory for HSV and VZV, Medical Center-University of Freiburg, Freiburg, Germany
  6. 6Institute for Medical Informatics, Biometry and Epidemiology (IMIBE), University Hospital Essen, University of Duisburg-Essen, Essen, Germany
  7. 7West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
  8. 8Department of Medicine II (Gastroenterology, Hepatology, Endocrinology and Infectious Diseases), Freiburg University Medical Centre, Faculty of Medicine, University of Freiburg, Freiburg, Germany
  1. Correspondence to Dr Dorothea Dehnen; dorothea.dehnen{at}


Introduction Shingrix, an effective adjuvanted, recombinant herpes zoster vaccine (RZV), has been available since 2018. Immunocompromised patients are known to be predisposed to vaccine failure. In-vitro testing of immunological surrogates of vaccine protection could be instrumental for monitoring vaccination success. So far, no test procedure is available for vaccine responses to RZV that could be used on a routine basis.

Methods and analysis This is a single-centre, three-arm, parallel, longitudinal cohort study aspiring to recruit a total of 308 patients (103 with a liver cirrhosis Child A/B, 103 after liver transplantation (both ≥50 years), 102 immunocompetent patients (60–70 years)). Blood samples will be taken at seven data collection points to determine varicella zoster virus (VZV) and glycoprotein E (gE)-specific IgG and T cell responses. The primary study outcome is to measure and compare responses after vaccination with RZV depending on the type and degree of immunosuppression using gE-specific antibody detection assays. As a secondary outcome, first, the gE-specific CD4+ T cell response of the three cohorts will be compared and, second, the gE-VZV antibody levels will be compared with the severity of possible vaccination reactions. The tertiary outcome is a potential association between VZV immune responses and clinical protection against shingles.

Ethics and dissemination Ethical approval was issued on 07/11/2022 by the Ethics Committee Essen, Germany (number 22-10805-BO). Findings will be published in peer-reviewed open-access journals and presented at local, national and international conferences.

Trial registration number German Clinical Trials Registry (number DRKS00030683).

  • hepatobiliary disease
  • immunology
  • general medicine (see internal medicine)
  • virology
  • transplant medicine

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  • This prospective study includes an immunocompetent control group allowing a comparison and assessment of potential immunological correlates of vaccine protection.

  • Successful patient recruitment is ensured by combining the appointments for titre determination with the regular appointments of patients with liver cirrhosis and post-liver transplantation patients in the outpatient liver clinic of University Hospital Essen, Germany.

  • A study nurse will perform home visit for patients who become immobile during the study period. This will ensure completeness of the data collection and reduce the dropout rate.

  • A placebo control group is not included.


Patients after solid organ transplantation are at an increased risk of infectious diseases due to drug-induced immunosuppression. Also, underlying (chronic) conditions, such as liver cirrhosis, most commonly due to hepatitis B or C and alcohol,1 lead to an immunodeficient state in which the immune system’s ability to fight infectious diseases is compromised due to cirrhosis-associated immune dysfunction.2–5 The cause for the immune dysfunction is multifactorial: damage to the reticuloendothelial system affecting the organ’s immune surveillance function,4 5 and an increase in serum levels of proinflammatory cytokines due to systemic inflammation.4 Thus, cirrhosis-associated immune dysfunction adversely affects the immune system, altering both innate and acquired immunity.6 This imminent threat also applies to herpes zoster (HZ, also termed shingles) which results from the reactivation of varicella zoster virus (VZV) replication in sensory ganglion cells,7 causing a painful, dermatome-related skin inflammation with vesicles. Especially under immunosuppression, severe, possibly life-threatening complications can occur.7 The incidence rate of HZ is significantly higher in immunocompromised individuals, such as liver transplant recipients (22.7 cases/1000 person-years (PY)),8 than in the general population (6.7 cases/1000 PY).9 Twenty per cent of patients with HZ after liver transplantation (LTx) suffer from disseminated shingles involving several dermatomes, visceral organs or the central nervous system, postherpetic neuralgia or cranial nerve damage.8

Since December 2018, the German Standing Committee on Vaccination (Ständige Impfkommission: STIKO) has recommended the adjuvanted HZ subunit inactivated vaccine (Shingrix) for people over 60 years of age (termed standard vaccination: vaccinations recommended for the general adult population) and for patients over 50 years of age with chronic underlying diseases associated with an increased risk of shingles such as diabetes mellitus or immunosuppressed patients (termed indicated vaccination).10 11 The vaccination itself is standard treatment.12

Recommendations are also being made at the international level to vaccinate immunosuppressed people at an early stage. The US Center for Disease Control and Prevention recommends vaccination from the age of 19 years,13 the European Centre for Disease Prevention and Control from the age of 18 years.14 The approved vaccination schedule recommends two vaccinations within the space of 2–6 months. Zoster vaccination is a preventive measure to protect against clinical consequences of VZV reactivation.2 The adjuvanted HZ subunit vaccine (recombinant zoster vaccine: RZV) consists of the recombinant VZV glycoprotein E (gE) and the AS01B adjuvant. gE is part of the VZV virion and is abundantly displayed on VZV-infected cells15; VZV gE antigen elicits neutralising antibodies and triggers vigorous CD4+ T cell responses.16

In immunocompetent individuals, the RZV has been shown to elicit robust IgG and CD4+ T cell responses associated with strong and long-lasting immune protection.17 18 Immunogenicity of RZVs was also documented in several groups of immunocompromised patients including adults with an HIV infection,19 after renal transplantation20 21 or adult patients after autologous stem cell transplantation.22 23 Immunogenicity and potential correlates of RZV vaccine-induced immune protection are not known in patients with an increased risk of shingles due to an impaired immune function of the liver or post-LTx. Reliable serological correlates of protection exist for some vaccinations (eg, hepatitis B),24–26 but this is not the case for many other vaccinations (eg, pertussis).24 In the pivotal studies of RZV (ZOE-50/70),17 18 a proprietary anti-gE enzyme-linked immunosorbent assay (ELISA) using a recombinant VZV gE antigen was used to determine anti-gE antibody levels. However, this ELISA was a laboratory developed test provided by the vaccine supplier which is not generally available. Conventional ELISAs using lysates or antigen preparations of VZV-infected cells do not determine antibodies that recognise gE, the antigen administered in the RZV. In order to measure the humoral immune response boosted by vaccination with RZV, a selective detection method is needed that especially measures anti-gE antibodies. For this purpose, we developed and validated two different gE-specific antibody detection methods at the Institute of Virology, University Hospital Freiburg: a gE-luciferase precipitation in-house assay analogous to Cohen et al27 and a gE ELISA, which is a modification of the assay described in Cunningham et al.28 Both assays will be used in the study.

The magnitude and duration of T cell and antibody responses are considered important for maintaining effective immune memory and protection against shingles.29–31 The immunogenicity results of the ZOE-50/70 studies showed a 24.6-fold28 increase in median gE-specific CD4+ T cell frequency 4 weeks after the second vaccination compared with baseline, and sustained gE-specific CD4+ T cell immunity was demonstrated in the interim analysis by Boutry et al.32 Immune responses were determined using VZV peptide pools to characterise virus-specific immunodominant epitopes. An independent study confirmed that vaccination with the RZV enhances the CD4+ T cell response against gE.33

Our study aims to quantify the emergence of VZV-specific CD4+ T cells in immunocompromised patients compared with a healthy control group after vaccination with the RZV. To the best of our knowledge, direct comparative studies on humoral and cellular immune response after vaccination with the RZV in patients with varying degrees of immunosuppression, especially in those with liver cirrhosis or after LTx, are not available. Thus, no serological parameters for the responsiveness of the immune system depending on the type and severity of immunosuppression have been investigated in these patient collectives so far. Reliable gE-specific antibody or T cell assays could provide valuable insights into the humoral and cellular immune response after vaccination and identify suitable correlates of protection which could be applied in the context of routine care of immunocompromised patients. Such data could provide the rational basis for future studies to investigate whether and when further vaccine doses might be necessary.



This single-centre, three-arm, parallel cohort study with a 1:1:1 allocation ratio is planned to take place between January 2023 and September 2028. Recruitment of primary care physicians and initial patients from the outpatient liver clinic began in January 2023. The study will include 103 patients with a secondary non-drug immunodeficiency due to chronic liver failure in the context of liver cirrhosis (stage Child A or B), 103 patients with a secondary drug-related immunodeficiency due to severe drug-related immunosuppression after LTx (both groups aged over 50 years) and 102 immunocompetent individuals who will receive the vaccination as a standard vaccination (age 60–70 years) (n=308) (see figure 1). The study design precludes randomisation.

Figure 1

Recruitment flow chart. (Figure made in*Major diseases: rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, chronic kidney disease, chronic obstructive pulmonary disease, bronchial asthma, diabetes mellitus, congenital or acquired immunodeficiency or immunosuppression/medical immunosuppression (eg, oral or intravenous cortisone, methotrexate, leflunomide), HIV infection, chronic alcohol abuse, chronic liver disease or status post-hepatitis. GP, general practitioner; HLA, human leucocyte antigen; VZV, varicella zoster virus.

The vaccination is part of the daily practice routine in Germany and is standard treatment. The STIKO vaccination recommendation is covered by statutory health insurance since March 2019.12 Since this is the daily routine of a European Medicines Agency-approved vaccine and a clinically indicated vaccination, the subjects will not receive an intervention in the strict sense. Thus, the study does not fulfil the criteria of an interventional study and is governed by the German Medical Professional Code.34 35


Study setting and characteristics of participants

This single-centre study is being conducted at the University Hospital in Essen, Germany. Each patient with chronic liver failure or with a liver transplant who meets the inclusion and exclusion criteria is being currently contacted (by phone, letter, personally) by the medical assistant, study nurse or doctors of the outpatient clinic of the Department of Gastroenterology and Hepatology. Patients in the control group are being recruited via the Institute of General Practice, Medical Faculty, University of Duisburg-Essen, Essen, or contacted by their general practitioner (GP) and their medical assistants, all of whom are part of the educational and research practice network of the above-mentioned institute. All persons enrolled in the study must provide full written informed consent and are required to complete a baseline screening questionnaire to assess their eligibility.

Inclusion and exclusion criteria

Inclusion criteria are:

Groups 1 and 2 (liver group)

  1. Age ≥50 years.

  2. Patients with liver cirrhosis Child A or B or who have undergone LTx.

Immunocompetent control group

  1. Patients without a chronic disease mentioned in the exclusion criteria and without drug-induced immunosuppression.

  2. Age 60–70 years.

All groups

Consent given by the patient or legal representative for vaccination and blood draw.

Exclusion criteria are:

  1. Negative VZV serostatus.

  2. Previous vaccination with Shingrix or Zostavax.

  3. Acute liver failure.

  4. Drug-induced immunosuppression in patients with chronic liver cirrhosis.

  5. LTx less than 6 months ago.

  6. Insufficient knowledge of the German language or inability to translate.

  7. Allergy to one of the components of RZV.

  8. Estimated life expectancy <6 months.

  9. Pregnancy.

  10. Known HIV infection.

  11. Active malignant tumour.

  12. Chemotherapy or antibody therapy in the last 12 months.

Additionally, in the immunocompetent control group, all diseases which, depending on the severity of the disease, can be associated with a more or less pronounced impaired immune response to vaccinations.

  1. Rheumatoid arthritis.

  2. Systemic lupus erythematosus.

  3. Inflammatory bowel disease.

  4. Chronic kidney disease (estimated glomerular filtration rate (eGFR) <45 mL/min).

  5. Chronic obstructive pulmonary disease.

  6. Bronchial asthma.

  7. Diabetes mellitus with an HbA1c >7.5%.

  8. Congenital or acquired immunodeficiency or immunosuppression/medical immunosuppression (eg, systemic cortisone therapy, methotrexate, leflunomide).

  9. Chronic alcohol addiction.

  10. Chronic liver disease or status post-hepatitis B/C virus or autoimmune hepatitis.

Intervention description

gE-specific antibody detection methods

The gE-specific luciferase assay uses a luciferase immunoprecipitation system (LIPS), which was described as particularly sensitive compared with other methods (fluorescent-antibody-to-membrane-antigen, glycoprotein ELISA, viral capsid antigen ELISA).27 In this assay, the gE ectodomain fused to Renilla luciferase is transiently expressed in HeLa cells, the lysate of which is incubated with patient serum. Immune complexes formed are precipitated using protein G Sepharose. The amount of specific anti-gE antibodies is determined by luciferase activity after washing the precipitated material. Due to the extremely sensitive luciferase detection, this procedure detects very low anti-gE antibody amounts (data not shown). This is of critical importance to include all eligible study participants and identify non-eligible individuals who need to receive varicella vaccination instead, depending on their immune status.

As the LIPS assay is not ideal for high throughput testing and exact quantification, a gE-specific ELISA based on the method described28 was developed. For this purpose, recombinant gE was obtained from Virion/Serion, Wuerzburg, Germany. gE antibody concentrations are calculated on the basis of a reference standard curve using Varitect (Biotest Pharma, Dreieich, Germany). The cut-off for the analysis was set at 88 arbitrary units (AU: greyzone 88–155 AU).

Humoral immunity

All three cohorts will receive their first vaccine dose with the RZV (t0) by their co-treating GP after confirmation of a positive VZV IgG serostatus. Patients who have undergone LTx will receive the first vaccination with the RZV within 6 months after transplantation (t0) in accordance with the STIKO recommendations. Pre-vaccination and post-vaccination samples will be tested using the in-house gE ELISA and, for comparison, with a standard VZV IgG ELISA (Virion/Serion, Wuerzburg, Germany). Pre-vaccination and post-vaccination samples with greyzone or negative results will be additionally tested with the most sensitive LIPS assay to identify truly negative sera. This is instrumental for the approach since this group of patients will be considered for receiving the live-attenuated varicella vaccine and must be excluded from the study, while individuals identified as seropositive in the LIPS assay will be included in the study. The first titre control will be performed in probands of all three study arms prior to the second vaccination (t1). Four to 6 weeks after the second vaccination, another titre control will take place (t2). Further titre checks are scheduled 6 (t3), 12 (t4), 36 (t5) and 60 (t6) months after the second vaccination with a window of 3 weeks if necessary. For details, see table 1 and figure 2.

Figure 2

Workflow flow chart. (Figure made in

Table 1

Research timeline for each participant

T cell immunity

VZV-specific CD4+ T cell responses will be analysed by flow cytometry using major histocompatibility complex II tetramers or VZV peptide pools. Participants will be human leucocyte antigen (HLA)-typed for class II (DRB1/DQB1/DPB1) alleles. For details, see table 1. All blood samples will be centrifuged and frozen until analysis. Upon arrival, peripheral blood mononuclear cells (PBMCs) will be isolated by Biocoll separation using Leucosep tubes. After centrifugation and washing of the cells, PBMC will be resuspended in fetal calf serum, aliquoted in 10% dimethyl sulfoxide and frozen for 3–4 weeks until analysis. The analyses of CD4+ responses will be performed as previously described36–38 by using tetramers recognising defined HLA class II alleles based on HLA typing of the study participants. Additionally, PBMC will be stimulated with VZV gE as well as control peptide mixes (JPT) to determine CD4 T cell responses before and after vaccination (see table 1 and figure 2).

Other laboratory and clinical parameters

To determine the current Child-Pugh score and the Model for End-Stage Liver Disease score, the current laboratory values (liver enzymes including glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, gamma-glutamyl transferase, alkaline phosphatase, coagulation (international normalised ratio) and kidney retention parameters including creatinine and eGFR as well as albumin and bilirubin in serum), sonographic signs of ascites and the hepatic encephalopathy grades39 will be recorded during routine visits to the outpatient clinic of the liver cirrhosis cohort, which are scheduled at the titre measurement appointments t0–t6. For details, see table 1.


Furthermore, all patients included in the study will receive a non-standardised questionnaire at each of the seven data collection points (t0–t6) asking about typical symptoms of the disease and possible vaccination reactions like local reaction at the injection site (eg, redness, swelling, pain) or systemic reactions (eg, general weakness, fever, headache, swelling of lymph nodes, chills).17 We will categorise the severity of the vaccination reaction into mild-to-moderate and serious adverse events, as in the pivotal studies.17 For details, see the Adverse event reporting and harms section.

In case of symptoms suggestive of HZ, a smear of the efflorescence will be sent to the Institute of Virology of the University Hospital Freiburg, Germany.

Participant timeline


Main outcome measures
Primary outcome measure

Comparison of the mean VZV antibody titre levels of patients with liver cirrhosis and those after LTx older than 50 years with the mean VZV antibody titre levels of immunocompetent individuals aged between 60 and 70 years from the control group.

Secondary outcome measures
  1. Comparison of the mean gE-specific CD4+ T cell frequencies in patients with liver cirrhosis and after LTx older than 50 years with those of immunocompetent patients aged 60–70 years from the control group.

  2. Comparison of the severity of vaccination reactions with the titre level after vaccination with RZV.

Tertiary outcome measure

Comparison of the number of vaccine breakthroughs, that is, the incidence of (severe) HZ infections confirmed by VZV-PCR, with titre level.


Adverse event reporting and harms

The risk of venous blood sampling is minimal. Pain or bruising may occur during or after the blood draw. In very rare cases, blood collection may result in inflammation of the puncture site (thrombophlebitis) or nerve injury.

There is a risk of vaccine reaction with transient symptoms such as local reactions (pain at the injection site, redness and swelling) and systemic reactions (fever, fatigue, myalgia and headache) noted in the first 7 days after vaccination and reported by 84.4% of those vaccinated.17 Of those vaccinated, 1.1% had serious adverse events (hypotension with syncope, mononeuritis, neurosensory deafness and musculoskeletal chest pain) within the first 30 days.17 Suspected cases of vaccination complication will be reported to the Paul-Ehrlich-Institute, the national institution responsible for vaccine pharmacovigilance.

Allergic reactions to components of the vaccine may occur in the first 15–30 min. Therefore, patients will be monitored for 15–30 min after vaccination.

The German Consulting Laboratory for HSV and VZV, Medical Center-University Hospital Freiburg, will carry out the titre measurements in compliance with the applicable safety guidelines.

The study investigator will rate the severity of each adverse event and report all serious and non-serious adverse events in the electronic case report form. The study investigator will also rate the underlying association between the serious adverse events and the study intervention. The following termination criteria were defined: severe allergic reaction to the RZV, occurrence of the above-mentioned serious events after vaccination17 as well as (in terms of localisation or extent) HZ within 1 month after the first or second vaccination, and occurrence of hepatic complications in the sense of acute to chronic liver failure.

Sample size calculation

Data from the vaccine approval studies ZOE-50 and ZOE-7017 18 showed mean VZV antibody titre levels in immunocompetent individuals of 52 376.6 mIU/mL 1 month after the second vaccination, with an SD of 35 996.96 mIU/mL. In their study, Vink et al40 observed that the VZV antibody titre level in renal transplant recipients 1 month after the second vaccination was on average at least 60% lower than in immunocompetent individuals. We consider a reduced titre increase of more than 30% in either group 1 or group 2, that is, a difference of more than 15 713.0 mlU/mL, as clinically relevant. The expected variance is based on the distribution in the approval study.17 18 40 An estimated loss to follow-up of 25% is included in our calculation.

Our case number calculation for our primary research aim therefore resulted in a required total study population of 308 subjects (n=102–103 per group) to detect a statistically significant difference of 30% (ie, 15 713.0 mlU/mL) or more in a group comparison by an analysis of variance (ANOVA) with a power of 80% and an alpha of 5%. The sample size calculation was performed with G-Power V.3.1 (and reviewed by a statistician from the Institute for Medical Informatics, Biometry and Epidemiology, Essen, Germany).

Plans to promote participant retention and complete follow-up

The participants will benefit from the study as they will receive detailed written and verbal information about a vaccination against HZ. For patients with liver cirrhosis and those after LTx, the appointments for the titre check (from t2) will be combined with the regular appointments in the liver outpatient clinic of the University Hospital Essen, Germany. If patients are not able to visit the outpatient clinic in person, they will be asked to visit their GP to provide a blood sample for the titre check. For this purpose, we will contact the respective GPs by telephone and ask them to draw the blood sample. If this is not possible, a study nurse will visit the patient at home to draw the blood sample. For the patients in the control group, the research practices (n=15–25 practices) of the Institute of General Practice, Essen and the Institute itself will carry out the titre controls. For this purpose, each practice will receive pre-labelled tubes and a stamped return envelope. The medical assistants of the practices will receive compensation in the amount of €60 per enrolled patient for the recruitment, vaccination and blood collection. The participants of immunocompetent control group will also receive an expense allowance totalling €60. Participants who wish to withdraw from the study or do not receive the second dose of the vaccine will be excluded from the study and will be asked to complete an end-of-study visit. If they do not object, the data collected to date will remain in the study database and will be included in the data analysis.

Data management

The principal investigator (Institute of General Practice, University Hospital Essen, Germany) will use an electronic case report form to record all study data. The lead physician will retain, in accordance with applicable regulations, all source documents, defined as any original document or item that allows evidence of the existence or accuracy of data or facts collected during the study. To ensure the dual control principle, all data from the questionnaires will be entered twice by two different individuals. A software tool will be used by a third person to check the two datasets (double entry) for consistency. If the data differ, the third person will determine the correct data from the questionnaires. If answers in the questionnaires do not allow a clear assignment, a consensual discussion will decide which entry will be made. All participant data will be kept in locked file cabinets to which only the principal investigator has access. Pseudonymisation of all data collected will be ensured through coding and will be traceable. All files containing names or other personal identifiers, such as consent forms, will be kept separate from the pseudonymised data. All blood samples for T cell analysis and HLA typing will be stored in the biobank of the Institute for Virology, University Hospital Essen for later analysis.

Statistical methods and data analysis plan

Statistical methods for primary and secondary outcomes

For our first research aim, the mean values of the vaccination titres at the time point t2 (4–6 weeks after the second vaccination) will be compared (F-test for ANOVA with subsequent post hoc t-tests). A special focus is placed on this time point, as this is when the VZV antibody titre level was highest in the pivotal trials and therefore reflects the vaccination response. Consequently, it will be analysed whether the vaccination titres at t2 differ significantly (p<0.05 for ANOVA and p<0.017 for post hoc t-tests to adjust for multiple testing with the Bonferroni correction) in the three groups. In the near future, when a titre value (cut-off value) reflecting sufficient immunity according to the assay is defined, it will be investigated whether these cut-off values were reached in all three groups at t2 (p<0.05). This will be analysed with a logistic regression or Χ2 test. The mean values of the vaccination titre at the other time points t1, t3, t4 (t5 and t6 serve as long-term controls) will be described descriptively. Subsequently, the mean values of the titre values of the three patient groups at t3 and t4 (with t5 and t6 serving as long-term controls) will be compared in order to assess the extent of an assumed drop in titre in the further course of the study within the three groups.

The mean values of the T cell frequency results at the t2 (4–6 weeks after second vaccination) will be compared (ANOVA with subsequent post hoc t-tests). The mean values of the T cell frequency results of the three groups at the other time points will be described descriptively, as will possible associations of titre levels with vaccination breakthroughs (occurrence of HZ) and the severity of vaccination reactions.

Since no explicit assumption on the distributions of the vaccination titres and T cell frequencies can be made in advance, we will examine these before the statistical analyses and, in case of a non-normal distribution, we will perform an appropriate transformation of titres and T cell frequency values for all non-descriptive analyses, for example, with the natural logarithm.

Possible confounders

Immune senescence can be a possible confounder. Thus, comparability appears to be much more difficult when comparing young patients with drug-induced immunosuppression with older patients without immunosuppression but with age-related immune senescence. Immune senescence is understood as an age-related decline in immunological competence and the associated progressive decrease in immune response at the humoral and cellular levels.41 42 Eliminating confounding due to age by matching participants in the three groups during the recruitment phase is likely not feasible, since this would restrict the liver groups to an age of 60 years and above. Therefore, we plan to control confounding due to age statistically in our analyses and perform regression analyses as sensitivity analyses for the primary and secondary research aim by adjusting for age. Gender will be included as a second covariate.23

No other relevant possible confounding factors are expected to have an impact on the dependent variables.

Patient and public involvement

No patients were involved in the development of the research questions. The results of the titre controls and the other laboratory parameters will be communicated to the patients as soon as they are available.


Ethics and dissemination

Ethical approval was issued on 07/11/2022 by the Ethics Committee Essen, Germany under the number 22-10805-BO. Findings will be disseminated initially to the impacted community, then by publication in scientific journals and at international congresses.


The study is registered with DRKS under the number DRKS00030683. Informed consent will be obtained from all participants in this study.

Ethics statements

Patient consent for publication


We acknowledge financial support by the University Hospital Essen Foundation (Stiftung Universitätsmedizin Essen), Germany; the Volker-Homann-Stiftung Freiburg, Germany; and the Open Access Publication Fund of the University of Duisburg-Essen, Germany; as well as endorsements by the North Rhine Association of Statutory Health Insurance Physicians (Kassenärztliche Vereinigung Nordrhein). The German Consulting Laboratory for HSV and VZV is supported in part by the Federal Ministry of Health, Berlin (Germany).



  • Contributors SV-R co-developed the concept and design of the study, helped raise funds, coauthored the draft manuscript and made revisions. JR-A contributed to the study concept and design and assisted in manuscript revision. SV contributed to the study concept and design, will raise funds for the T cell analysis and assisted in manuscript revision. HH contributed to the study concept and design and assisted in manuscript revision. BB carried out the case number planning, conducted the scheduling of the statistical analysis and assisted in manuscript revision. DH contributed to the study concept and design and assisted in manuscript revision. E-MH carried out the case number planning, conducted the scheduling of the statistical analysis and assisted in manuscript revision. JidS contributed to the study concept and design and assisted in manuscript revision. AH contributed to the study concept and design and assisted in manuscript revision. KW contributed to the study concept and design. SB contributed to the study concept and design and assisted in manuscript revision. MT contributed to the study concept and design and assisted in manuscript revision. TB contributed to the study concept and design. DD is the principal investigator who conceived the study, obtained funding, led the proposal and protocol development, drafted the manuscript preparation and headed its revision. All authors read and approved the final manuscript.

  • Funding This work was supported financially by grants of the University Hospital Essen Foundation, Germany (Stiftung Universitätsmedizin Essen, grant number: 2022 4699 105) to DD and by grants of the Volker-Homann-Stiftung, Freiburg, Germany (grant number: N/A) to DH. Furthermore, this study is supported by institutional funds of the Institute of General Practice, Medical Faculty, University of Duisburg-Essen, Essen (grant number: N/A) and the Institute of Virology at the Freiburg University Medical Centre and the Faculty of Medicine, University of Freiburg, Germany (grant number: N/A).

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.