Characterization of Humoral and Cellular Immunity for Tick-borne Encephalitis (TBE) Vaccination in Allogeneic Blood and Marrow Graft Recipients: a Pilot Study
Phase: PHASE2
Conditions: Tick Borne Encephalitis
Sponsor: Medical University of Vienna

Immunity_TBE 
Department of Internal Medicine I, Division of Infectious Diseases and Tropical Medicine 
EUDRACT Nr 2011-002928-41 
 
Date 24.08.2015 
 
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CLINICAL STUDY PROTOCOL 
 
 
TITLE 
 
Characterization of humoral and cellular immunity for tick-borne encephalitis (TBE) 
vaccination in allogeneic blood and marrow graft recipients: a pilot study 
 
SHORT TITLE 
Immunity for TBE vaccination in allogeneic blood and marrow graft recipients 
 
PROTOCOL No. 4 
 
EUDRA CT No. 2011-002928-41 
 
 
Version 4.0 / 24.08.2015 
 
 
 
Confidentiality Statement 
This document is confidential. Receipt of this document constitutes agreement by the recipient that 
no unpublished information contained herein shall be published or disclosed without prior written 
approval, except that this document may be disclosed to the appropriate Institutional Review 
Board/Independent Ethics Committee under the condition that they keep confidential. It is 
understood that this information will not be disclosed to others without written authorization, 
except to the extent necessary to obtain informed consent from those persons to whom the study 
drug may be administered. 
 
 

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Test drug (IMP) and 
Pharmaceutical Company  
FSME IMMUN (FSME-Virus inaktiviert,Neudörfl strain) 2,4µg; PFIZER 
Protocol authors 
Christina Forstner, MD;1 Heinz Burgmann, MD1 
1Department of Internal Medicine I, Division of Infectious Diseases and 
Tropical Medicine, Medical University of Vienna, Währinger Gürtel 18-
20, 1090 Vienna 
Investigators 
Christina Forstner, MD;1 Heinz Burgmann, MD;1 Nicole Harrison, MD;1 
Hildegard Greinix, MD;2 Peter Kalhs, MD;2 Zoya Kuzmina, MD;2 Franz 
Xaver Heinz, PhD;3 Karin Stiasny, PhD;3 Judith Aberle, MD;3 Alexandra 
Graf, MD;4 Katharina Grabmeier-Pfistershammer, MD5 
1Department of Internal Medicine I, Division of Infectious Diseases and 
Tropical Medicine, Medical University of Vienna 
2Department of Internal Medicine I, Division of Bone Marrow 
Transplantation, Medical University of Vienna 
3Department of Virology, Medical University of Vienna 
4Institute of Medical Statistics, Medical University of Vienna 
5Department of Dermatology, Medical University of Vienna 
Document type 
Clinical study protocol 
Study phase 
A prospective open-label phase II pilot study 
Date 
24.08.2015 
Number of pages 
44 

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1. SPONSOR, INVESTIGATOR, MONITOR 
 
Sponsor (AMG §§ 2a, 31,32) 
 
Department of Internal Medicine I, Division of Infectious Diseases and Tropical Medicine Medical 
University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria 
 
Investigator (AMG §§ 2a, 35,36) 
 
 
Ao. Univ.-Prof. Dr. Heinz Burgmann, Department of Internal Medicine I, Division of Infectious 
Diseases, Medical University of Vienna, Austria 
 
Monitor (AMG §§ 2a, 33,34) 
 
 
Priv.Doz. Dr. Michael Ramharter, Department of Internal Medicine I, Division of Infectious Diseases, 
Medical University of Vienna 
Dr. Wolfgang Pöppl, Department of Dermatology, Medical University of Vienna 
 
Statistician 
 
 
Dr. MMag. Alexandra Graf, Institute for Medical Statistics, Medical University of Vienna 
 
 

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2. PROTOCOL SYNOPSIS 
TITLE 
Characterization of humoral and cellular immunity for tick-borne 
encephalitis (TBE) vaccination in allogeneic blood and marrow graft 
recipients: a pilot study 
OBJECTIVES 
Primary Objective 
• 
To assess the humoral immunogenicity to a TBE vaccination in 
allogeneic blood and marrow graft (HSCT) recipients compared to 
healthy volunteers without previous TBE vaccination, by measuring 
the quantitative antibody levels using neutralization test (NT) and 
enzyme-linked immunosorbent assay test (ELISA)  
Secondary Objectives 
• 
To assess cellular immunogenicity of the TBE vaccination in 
allogeneic HSCT recipients, by measuring the lymphocyte 
proliferation and cytokine levels after in vitro stimulation with 
peripheral blood mononuclear cells with whole aluminium 
hydroxide-free and human albumin-free TBE antigen  
• 
To assess the immune status in HSCT recipients prior to and after 
vaccination, as measured by quantitative immunoglobulin levels 
and by immunofluorescence staining of peripheral blood T and B 
lymphocytes and flow cytometry analyses 
DESIGN / PHASE 
Prospective, open-label phase II study. 
STUDY PLANNED DURATION First patient 
First visit 
07/ 
2014 
 
Last patient 
First visit 
12/ 
2016 
 
Last patient 
Last visit 
12/ 
2017 
 
CENTER(S) 
/ COUNTRY(IES)  
Single center study of the Medical University of Vienna in Austria 
PATIENTS / GROUPS 
26 HSCT patients in the study group 
26 healthy volunteers in the control group 
INCLUSION CRITERIA 
• 
Adult patients ≥18 years who 
- 
had undergone an allogeneic HSCT 11 to 13 months ago or 
- 
healthy volunteers without previous TBE vaccination 
EXCLUSION CRITERIA 
• 
Previous TBE vaccination following HSCT 
• 
HSCT patients with extremely severe acute graft versus host 
disease (receiving prednisone >0.5 mg/kg bodyweight as part of a 
combination therapy or a three agent immunosuppressive 
treatment)  
• 
Previous TBE virus infection, previous dengue virus infection or 
vaccination against yellow fever or Japanese encephalitis 
• 
Any acute febrile illness in the 2 weeks prior to or at the time of 
enrolment 
• 
A history of severe allergic reactions or anaphylaxis after 
vaccination 
• 
If female, are pregnant or lactating 
• 
If belonging to the healthy control group, are immunosuppressed 
• 
Relapse of underlying malignant disease 
STUDY PERIODS 
• The active phase of the study for each participant is 44-56 weeks 
• No additional controls are needed 

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• The total duration of the study is estimated to be 4 years 
STUDY DRUG 
TBE vaccine – FSME IMMUN 0.5 ml (2.4 µg) 
COMPARATIVE DRUG  
No 
EFFICACY ENDPOINTS 
Number of subjects with antibody response to TBE vaccination 
associated with protection 28 days after the second and third 
vaccination 
TOLERABILITY / SAFETY 
ENDPOINTS 
Number of subjects with systemic reactions after vaccination 
PHARMACOKINETIC / 
PHARMACODYNAMIC 
ENDPOINTS 
No 
QUALITY OF LIFE / 
PHARMACOECONOMIC  
ENDPOINTS 
No 
STATISTICAL METHODOLOGY  Primary Endpoint 
To assess humoral immunogenicity of the TBE vaccination in 
allogeneic HSCT recipients compared to healthy volunteers without 
previous TBE vaccination, the outcome of the neutralilzation test (NT) 
will be assessed four weeks after the second vaccination. Therefore, 
the number of subjects with NT titers against TBE virus >10 
(=seroconversion rate), assumed to be the threshold for antibody-
mediated protection, will be evaluated. 
Null and alternative hypotheses: 
H0: There is no statistical difference in the seroconversion rate between 
the study population and the control group 
H1: There is statistical difference in the seroconverion rate between the 
study population and the control group 
Type-I and -II errors – power:  
Alpha 0.05. Power 0.8. 
 
Sample size calculation 
The calculation of the sample size was performed using nQuery 6.1. 
The primary endpoint is the outcome of the NT against 4 weeks after 
the second vaccination (positive test: Yes/No). Because of deviation of 
the antibody concentrations from normal distribution (NT level <=10 
not detectable indicating a negative test), the outcome is reduced to a 
binary but clinically more relevant outcome (seroconversion rate: NT 
levels >10 indicates a positive test). Previously, in a study with heart 
transplant recipients a seroconversion rate of 35% was detected 
compared to 100% in the healthy control group. In our study we expect 
a seroconversion rate of at least 90% in the healthy control group. In 
the HSCT patients we expect a seroconversion not exceeding 50%. The 
calculation of the sample size was performed using the following 
assumption: Significance level alpha 0.05, Power 0.8, ratio of HSCT 
patients/healthy volunteers: 1/1. With a total number of 46 
participants (23 HSCT patients and 23 healthy volunteers) the assumed 
difference in the seroconversion rate between groups can be detected 
(using a two-sided Fisher’s Exact Test) with 80% Power. We added 10% 
to adjust for potential loss-to-follow-up which results in 26 HSCT 
patients and 26 healthy volunteers. 
 

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Statistical methodology 
The primary endpoint (outcome of NT 4 weeks after the second 
vaccination) will be analysed using Fisher’s Exact Test. For the 
seroconversion rates, 95% confidence intervals will be calculated. 
The secondary endpoints (NT 4 weeks after the third vaccination, ELISA 
after 
the 
second 
and 
third 
vaccination, 
cellular 
immunity, 
immunoglobulin levels, immune reconstitution and safety data) will be 
analysed using descriptive statistics. 
 
 
3. LIST OF ABBREVIATIONS 
 
AE = Adverse event 
CFSE = carboxyfluroescein diacetate succinimidyl ester 
CRF = Case Report Form 
ELISA = enzyme-linked immunosorbent assay 
FACS = fluorescence activated cell sorting 
GCP = Good Clinical Practice 
GVHD = graft-versus-host disease  
HSCT = blood and marrow transplantation 
ICH = International Conference on Harmonization  
NIH = National Institute of Health 
NT = neutralization test 
PBMCs = peripheral blood mononuclear cells 
PHA = phytohemagglutinin 
TBE = tick-borne encephalitis 
 
 

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4. TABLE OF CONTENTS 
 
CLINICAL STUDY PROTOCOL 
1 
1. SPONSOR, INVESTIGATOR, MONITOR 
3 
2. PROTOCOL SYNOPSIS 
4 
3. LIST OF ABBREVIATIONS 
6 
4. TABLE OF CONTENTS 
7 
TABLE 1. VISIT AND ASSESSMENT SCHEDULE 
10 
5. BACKGROUND INFORMATION 
11 
5.1 BACKGROUND 
11 
5.2 STUDY RATIONALE 
14 
6. STUDY OBJECTIVES 
14 
6.1 PRIMARY OBJECTIVE (HYPOTHESIS) 
14 
6.2 SECONDARY OBJECTIVES 
15 
7. STUDY DESIGN 
15 
7.1 STUDY POPULATION 
16 
7.1.1 SUBJECT POPULATION 
16 
7.1.2 INCLUSION CRITERIA 
16 
7.1.3 EXCLUSION CRITERIA 
16 
7.1.4 STUDY DURATION 
17 
7.1.5 WITHDRAWAL AND REPLACEMENT OF SUBJECTS 
17 
7.1.6 PREMATURE TERMINATION OF THE STUDY 
18 
8. METHODOLOGY 
18 
8.1 STUDY MEDICATION 
18 
8.1.1 DOSAGE AND ADMINISTRATION 
19 
8.1.2 STUDY-DRUG UP- AND DOWN TITRATION 
19 
8.1.3 STUDY DRUG INTERRUPTION OR DISCONTINUATION 
19 
8.1.4 STUDY-DRUG DELIVERY & DRUG STORAGE CONDITIONS 
19 

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8.1.5 STUDY DRUG PACKAGING AND LABELING 
20 
8.1.6 IMP ADMINISTRATION & HANDLING 
20 
8.1.7 DRUG ACCOUNTABILITY 
20 
8.1.8 PROCEDURES TO ASSESS SUBJECTS COMPLIANCE 
20 
8.1.9 CONCOMITANT MEDICATION 
20 
8.2 RANDOMIZATION 
20 
8.3 BLINDING 
20 
8.4 BENEFIT AND RISK ASSESSMENT 
20 
8.5 STUDY PROCEDURES 
21 
8.5.1 GENERAL RULES FOR TRIAL PROCEDURES 
21 
8.5.2 STUDY VISITS 
21 
8.5.2.1 VISIT 0/ SCREENING INVESTIGATION (DAY -28) 
21 
8.5.2.2. VISIT 1 /FIRST VACCINATION (DAY 0) 
22 
8.5.2.3. VISIT 2 /SECOND VACCINATION (DAY 28 ± 2 DAYS) 
23 
8.5.2.4. VISIT 3 /1. EVALUATION OF CELLULAR IMMUNITY (DAY 35, 7 ± 2 DAYS AFTER THE 
SECOND VACCINATION) 
23 
8.5.2.5. VISIT 4 /1. EVALUATION OF HUMORAL IMMUNITY (DAY 56, 28 ± 7 DAYS AFTER THE 
SECOND VACCINATION) 
23 
8.5.2.6. VISIT 5 /1. EVALUATION OF IMMUNE STATUS (DAY 84 ± 14 DAYS) 
23 
8.5.2.7. VISIT 6 /3. VACCINATION AND EVALUATION OF IMMUNE STATUS (DAY 270-365) 
23 
8.5.2.8. VISIT 7 /2. EVALUATION OF CELLULAR IMMUNITY (DAY 277-372, 7 ± 2 DAYS AFTER THE 
THIRD VACCINATION) 
24 
8.5.2.9. VISIT 8 /2. EVALUATION OF HUMORAL IMMUNITY (DAY 298-393, 28 ± 7 DAYS AFTER THE 
THIRD VACCINATION) 
24 
8.5.3 DETERMINATION OF THE HUMORAL AND CELLULAR IMMUNITY 
24 
8.5.3.1 DETERMINATION OF SERUM ANTIBODIES AGAINST VACCINATION ANTIGENS 
24 
8.5.3.2 DETERMINATION OF CELLULAR IMMUNE RESPONSE 
25 
8.5.3.3 DETERMINATION OF IMMUNE RECONSTITUTION IN HSCT RECIPIENTS 
26 
8.5.4 STUDY ENDPOINTS 
26 
8.5.4.1 PRIMARY STUDY ENDPOINT 
26 
8.5.4.2 SECONDARY STUDY END POINTS 
26 
9. SAFETY DEFINITIONS AND REPORTING REQUIREMENTS 
27 
9.1 ADVERSE EVENTS (AES) 
27 
9.1.1 SUMMARY OF KNOWN AND POTENTIAL RISKS OF THE STUDY DRUG 
27 
9.1.2 DEFINITION OF ADVERSE EVENTS 
27 
9.2 SERIOUS ADVERSE EVENTS (SAES) 
28 
9.2.1 HOSPITALIZATION – PROLONGATION OF EXISTING HOSPITALIZATION 
29 
9.2.2 SAES RELATED TO STUDY-MANDATED PROCEDURES 
29 
9.2.3 SUSPECTED UNEXPECTED SERIOUS ADVERSE REACTIONS (SUSARS) 
29 
9.2.4 PREGNANCY 
30 
9.3 SEVERITY OF ADVERSE EVENTS 
30 
9.4 RELATIONSHIP TO STUDY DRUG 
31 
9.5 REPORTING PROCEDURES 
32 

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9.5.1 REPORTING PROCEDURES FOR SAES 
32 
9.5.2 REPORTING PROCEDURES FOR SUSARS 
33 
9.5.3 ANNUAL SAFETY REPORT 
34 
10. FOLLOW-UP 
34 
11. STATISTICAL ANALYSIS 
34 
11.1 SAMPLE SIZE CONSIDERATIONS 
34 
11.2 RELEVANT PROTOCOL DEVIATIONS 
35 
11.3 ENDPOINTS ANALYSIS 
35 
11.4 MISSING, UNUSED AND SPURIOUS DATA 
35 
11.5 INTERIM ANALYSIS 
35 
11.6 SOFTWARE PROGRAM(S) 
35 
12. DOCUMENTATION AND DATA MANAGEMENT 
35 
12.1 DOCUMENTATION OF STUDY RESULTS 
35 
12.1.1 CASE REPORT FORM (CRF) 
36 
12.1.2 DATA COLLECTION 
36 
12.2 SAFEKEEPING 
36 
12.3 QUALITY CONTROL AND QUALITY ASSURANCE 
37 
12.3.1 PERIODIC MONITORING 
37 
12.3.2 AUDIT AND INSPECTIONS 
38 
12.4 REPORTING AND PUBLICATION 
38 
12.4.1 PUBLICATION OF STUDY RESULTS 
38 
13. ETHICAL AND LEGAL ASPECTS 
38 
13.1 INFORMED CONSENT OF SUBJECTS 
38 
13.2 ACKNOWLEDGEMENT / APPROVAL OF THE STUDY 
39 
13.2.1 CHANGES IN THE CONDUCT OF THE STUDY 
39 
13.3 INSURANCE 
40 
13.4 CONFIDENTIALITY 
40 
13.5 ETHICS AND GOOD CLINICAL PRACTICE (GCP) 
40 
14. APPENDICES 
41 
15. REFERENCES 
42 
 

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TABLE 1. VISIT AND ASSESSMENT SCHEDULE 
 
 
PERIODS 
Name 
SCREENING 
VACCINATION 
 
Duration 
 
44-56 weeks 
VISITS  
Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
 
Name 
Screening 
Visit 1/ 
1st  
Vaccination 
Visit 2/ 
2nd 
Vaccination 
Visit 3 
Visit 4 
Visit 5 
Visit 6/  
3rd 
Vaccination 
Visit 7 
Visit 8 
 
Time 
Day  
– 28 
Day 0 
Day 28 
(± 2 days) 
Day 35 
(7 ± 2 days 
after Visit 2)  
Day 56 
(28 ± 7 days 
after Visit 2) 
Day 84 
(± 14 days) 
Day 270-
365 
Day 277-372 
(7± 2 days 
after Visit 6) 
Day 298-393 
 (28 ± 7 days 
after Visit 6) 
Informed Consent 
X 
 
 
 
 
 
 
 
 
Inclusion / Exclusion Criteria 
X 
X 
 
 
 
 
 
 
 
Medical History 
X 
X 
 
 
 
 
 
 
 
Physical Examination 
X 
X 
X 
 
 
 
X 
 
 
Pregnancy Test 
X 
X 
X 
 
X 
X 
X 
 
X 
Blood Draw (Serology) 
X 
 
 
 
X 
 
 
 
X 
Blood Draw (Cellular immunity) 
X 
 
 
X 
 
 
 
X 
 
Blood Draw (Immune 
reconstitution + serum 
immunoglobulin levels) 
X 
 
 
 
 
X 
X 
 
 
Vaccination 
 
X 
X 
 
 
 
X 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
 

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5. BACKGROUND INFORMATION 
5.1  Background  
 
Allogeneic blood and marrow transplantation (HSCT) are increasingly employed in the 
treatment of hematological and oncological malignancies and are now standard procedures 
for selected diseases. During, immunosuppressive treatment is required to reduce the risk of 
transplant rejection and graft-versus-host disease (GVHD).  
Serious infections are common in the early phase following allogeneic HSCT because 
immune reactivity during the first month post-transplant is very low. Effective approaches 
toward vaccinating patients against common pathogens are being explored but are limited by 
poor levels of responsiveness. Protective immunity to diseases preventable by routine 
vaccination is lost following allogeneic HSCT. Adoptive transfer of immunity from donors to 
recipients after allogeneic transplantation is not sufficient to prevent this decline. However, 
vaccination practices after transplantation vary and data are sparse [1, 2]. 
Following transplantation, cytotoxic and phagocytic functions recover by day 100, but the 
more specialized function of T and B lymphocytes may remain impaired for a year or even 
longer. Deficiencies in cellular immunity are characterized by the inversion of CD4/CD8 
ratios, a decreased proliferative response to mitogens, and the development of anergy to 
recall antigens as measured by delayed-type hypersensitivity testing. The impact on humoral 
immunity 
consists 
of 
decreased 
levels 
of 
circulating 
immunoglobulins, 
impaired 
immunoglobulin class switching, and a loss of complexity in immunoglobulin gene 
rearrangement patterns. The efficacy of vaccination following HSCT is influenced by the time 
elapsed since transplantation, the nature of the hematopoietic graft, the use of serial 
immunization, the vaccine being used, and the immune status of the patient including the 
presence of chronic GVHD [3]. After one to three years after allogeneic HSCT patients 
without chronic GVHD experience complete immune regeneration whereas in patients with 
chronic GVHD immunodeficiency can remain for prolonged periods of time [4-6].  
Chronic GVHD continues to be a major problem in long-term survivors of allogeneic HSCT, 
affecting 35-70% of patients [7, 8]. The pathogenesis and clinical features of chronic GVHD 
resemble those of several autoimmune diseases such as systemic lupus erythematosus, 
Sjogren’s syndrome and scleroderma. Autoreactive T lymphocytes are an important effector 
mechanism with interferon gamma playing a central role in the increased collagen deposition 
that is a central histopathologic feature of chronic GVHD [9]. Since chronic GVHD only 
occurs in allogeneic HSCT recipients and can be prevented by T-cell depletion from the 
donor graft, donor T-cells responding to allogeneic antigens in the patient are of critical 

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importance for the development of chronic GVHD. Of note, donor B-cell responses to 
recipient HY antigens have been significantly associated with the development of chronic 
GVHD in the setting of gender mismatched HSCT.  
Chronic GVHD can affect the skin (depigmentation, poikiloderma, lichen planus-like features, 
sclerotic features, morphea-like features, lichen sclerosus-like features lichenoid papules), 
the liver (hepatitis), the lung (obliterative bronchiolitis), the oral mucosa (lichen-type features, 
hyperkeratotic plaques, restriction of mouth opening from sclerosis, ulcerations, atrophy of 
the mucosa, xerostomy), eyes (keratoconjunctivitis sicca), muscles and fibers (myositis, 
tendinitis, and fasciitis), and the esophagus (esophageal web, strictures or stenosis), 
respectively. The diagnosis of chronic GVHD requires the presence of at least one diagnostic 
clinical sign of chronic GVHD (e.g. poikiloderma) or the presence of at least one distinctive 
manifestation (e.g. keratoconjunctivitis sicca) confirmed by pertinent biopsy or other relevant 
tests (e.g. Schirmer test) in the same or another organ. According to the consensus of the 
National Institute of Health (NIH) in 2005 a new clinical scoring system (0-3) was introduced 
to assess the extent and severity of chronic GVHD for each organ or site at any given time, 
with 0 representing no involvement and 3 reflecting severe impairment [10]. For mild forms, 
no treatment is necessary. Extended disease requires specific immunosuppressive treatment 
using corticosteroids +/- cyclosporine, tacrolimus, and monoclonal antibodies such as 
basiliximab 
or 
tumor 
necrosis 
factor-alpha 
inhibitor 
as 
etanercept 
[11]. 
These 
immunosuppressive regimens may additionally impair patients’ response to vaccination. 
Therefore, infections have become increasingly important during GVHD therapy [12]. 
Immunosuppressive therapy is being improved continually and in most cases little is known 
about the impact of the more recently adopted regimens on durability of antibody response. 
Thus, 
studies 
on 
immune-response 
after 
vaccination 
in 
patients 
with 
different 
immunosuppressive regimes are still missing.  
Tick-borne encephalitis (TBE) is a disease of the central nervous system caused by a tick-
borne flavivirus infection. TBE can lead to severe neurological symptoms such as meningitis, 
meningoencephalitis, and meningoencephalomyelitis, which can result in death. There is no 
treatment, and prevention with the vaccine is the only intervention currently available. TBE is 
the most important arthropod-transmitted viral disease in Europe, and in some countries it 
represents a major public-health problem [13]. In Austria and the southern parts of Germany 
TBE is endemic and therefore it is assumed that the whole population living in those areas is 
at risk. In the pre-vaccination era, Austria had the highest recorded morbidity of TBE in 
Europe. The disease accounted for more than 50% of all viral meningoencephalitides in the 
eastern and southern parts of the country [14]. Flaviviruses are a large group of small, 
enveloped viruses responsible for a number of severe human diseases, including yellow 

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fever, Japanese encephalitis, dengue hemorrhagic fever and TBE. TBE virus particles are 
roughly spherical in shape, 40-50nm in diameter, and contain a core, 20-30nm in diameter. 
The genome consists of single-stranded positive-sense RNA with a relative molecular mass 
of about 4x106. Three structural proteins and the capsid, membrane and envelope proteins 
are all encoded by the viral genome. The envelope glycoprotein induces neutralizing and 
hemagglutination-inhibition antibodies and is the most important antigen for providing 
protection from disease.  
The first vaccine against TBE was prepared in 1941 in the brains of mice. Some 20 years 
later TBE vaccines derived from cell cultures (chicken embryo fibroblast cells) were 
developed and used for active immunization in humans in the former Soviet Union. Later, a 
purified, inactivated virus vaccine was developed which proved to be more immunogenic 
than previous TBE vaccines. The efficacy of these vaccines has been well documented. All 
the currently licensed TBE vaccines (Encepur children, Encepur Adults, and FSME-IMMUN 
"new") gave high seroconversion rates of over 87% in immunocompetent individuals. 
Although systemic and local adverse event were commonly reported, none were serious or 
life-threatening [15]. 
Following allogeneic HSCT, TBE vaccination should be performed as primary immunization 
in risk area with three vaccine-doses after the first nine to twelve months (CIII 
recommendation) [16].  
Adequacy of the immune response to a vaccine is frequently measured by the serum level of 
the specific antibody. Although seroconversion does indicate an immune response, it does 
not necessarily signify protection. While routine determination of antibody titers prior to 
vaccination is not recommended in all transplant recipients, post-vaccination determination of 
antibody levels is useful to monitor antibody response and protection, and assess the need 
for additional doses of the vaccine [1]. Successful vaccination can be easily monitored by the 
demonstration of specific immunoglobulin G-antibodies in the serum by enzyme-linked 
immunosorbent assay (ELISA). However, there is a potential for misleading results, because 
all flaviviruses are serologically related and infections or vaccinations with one flavivirus will 
give rise to antibodies that also react with all other flaviviruses in ELISAs. Vaccinations 
against yellow fever, Japanese encephalitis or infections with dengue viruses will thus result 
in cross-reactive but non-neutralizing antibodies yielding a positive result in TBE ELISAs [13]. 
Detectable neutralizing antibodies are generally viewed as a surrogate parameter of 
protection against TBE infection because their formation is of decisive importance for virus 
elimination and entails protection against the disease [17]. 

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Serological response to TBE vaccination has already been studied in different populations 
with decreased responsiveness to vaccines, the HIV-positive patients [18], in the elderly [19, 
20], and in heart transplant recipients [21], respectively.  
For pneumococcal polysaccharide vaccine, lack of responsiveness to vaccination in HSCT 
patients with chronic GVHD has been demonstrated [22]. But there is little information on the 
efficacy of the TBE vaccine in stem cell transplant recipients with chronic GVHD.  
 
5.2  Study rationale 
 
Patients undergoing HSCT experience a prolonged period of dysfunctional immunity 
associated with an increased risk of bacterial and viral infections. Systematic re-
immunization is necessary at appropriate time intervals following transplantation to re-
establish immunity [1]. TBE vaccination should be performed as primary immunization in risk 
area with three vaccine-doses nine to twelve months after allogeneic HSCT (CIII 
recommendation) [16]. However, immune response after vaccination might vary a lot among 
HSCT patients with and without chronic GVHD. In a study with immunosuppressed heart 
transplant recipients a seroconversion rate of 35% was detected compared to 100% in the 
healthy control group [21]. Similarly, we assume that the serum conversion rate after TBE re-
immunization is significantly reduced in HSCT recipients compared to an age-matched and 
sex-matched control group of healthy volunteers without previous TBE vaccination. 
Moreover, it is not clear whether TBE vaccination in HSCT patients with chronic GVHD has a 
protective effect at all.  
The results of the planned pilot study will provide first data to assess the efficacy of TBE 
vaccination in allogeneic HSCT recipients with and without chronic GVHD. These data will 
enable to develop new guidelines or optimize current guidelines for TBE vaccination in this 
special population at risk. It is expected that different guidelines for HSCT patients with and 
without chronic GVHD have to be established in the future. 
6. STUDY OBJECTIVES 
 
The aims of this study are to estimate the humoral and cellular immune responses after TBE 
vaccination in adult HSCT recipients. 
6.1  Primary Objective (Hypothesis) 
 
To assess the humoral immunogenicity to a TBE vaccination in allogeneic HSCT recipients 
compared to healthy volunteers without previous TBE vaccination, the quantitative antibody 

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levels will be measured using neutralilzation test (NT) and enzyme-linked immunosorbent 
assay test (ELISA). 
The null hypothesis has to be rejected that there is no difference in the seroconversion rate 
between the HSCT patients and the healthy volunteers after TBE vaccination. 
6.2  Secondary Objectives  
 
To assess cellular immunogenicity of the TBE vaccination in allogeneic HSCT recipients, T-
cell cytokine expression will be measured using flow-cytometry after in vitro stimulation with 
peripheral blood mononuclear cells with TBE antigen. 
To evaluate the immune status in HSCT recipients prior to and after vaccination, quantitative 
immunoglobulin levels will be measured and immunofluorescence staining of peripheral 
blood T and B lymphocytes and flow cytometry analyses will be performed. 
7. STUDY DESIGN 
 
A prospective open-label phase II pilot study will be performed. A total of 52 adult male or 
female subjects, 26 HSCT patients (study population) and 26 healthy volunteers (control 
group), will be enrolled in this clinical trial. Eight study visits per patient will be planed. 
Eligible patients will receive at least two TBE vaccinations (study visit 1 – day 0, study visit 2 
-1month after the first vaccination) with a total of two doses of the TBE vaccine FSME 
IMMUN®. Whenever possible, the patients will receive complete primary vaccination with a 
third dose of TBE vaccine FSME IMMUN® (study visit 9 - 12 months after the first 
vaccination). TBE vaccine will be injected intramuscularly in the Musculus deltoideus. 
FSME IMMUN® contains formalin-inactivated TBE virus. One dose (0.5 ml) contains 2.4 µg 
of antigen inactivated by formaldehyde. Human albumin is used as stabilizer and aluminium 
hydroxide is used as adjuvant. The vaccine contains traces of formaldehyde, protamine 
sulphate, neomycin and gentamicin.  
Serum samples for determination of antibody levels will be obtained immediately before the 
first (study visit 1), 4 weeks after the second vaccination (study visit 4), and four weeks after 
the third vaccination (study visit 8). Blood samples for the analysis of cellular immunity will be 
taken before the first (study visit 1), one week after the second vaccination (study visit 3) and 
one week after the third vaccination (study visit 7). Blood samples for determination of the 
immune reconstitution and the quantitative immunoglobulin levels will be obtained prior to the 
first (study visit 1) and third vaccination (study visit 6) and 12 weeks after the first vaccination 
(study visit 5) in HSCT recipients only.  
 

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7.1  Study population 
 
Adult patients ≥18 years will be recruited at the bone marrow transplant center of the Medical 
University of Vienna. Patients will be eligible if 12 months +/- 1 month had passed since the 
allogeneic HSCT and if the patient is willing to sign an informed consent. Basic demographic 
data including age, gender, conditioning regimen, type of the hematopoietic graft 
(sister/brother, unrelated donor, cord blood) or bone marrow transplant, intensity of 
immunosuppressive therapy, medical history including time of previous TBE vaccination 
before HSCT and present status of chronic GVHD according to the NIH criteria [10] will be 
obtained. If available (only possible for sister/brother donor) the current TBE vaccination 
status of the donors will be requested. As control group, non-immunocompromised adult 
volunteers ≥18 years (age-matched with a tolerance of +/-10 years and sex-matched to the 
study population) without previous TBE-vaccination will be recruited at the outpatient clinic of 
the department of infectious diseases and tropical medicine of the Medical University of 
Vienna. 
7.1.1 Subject population 
 
Study entry is defined as the date of signature of the study participant (subject) on the 
informed consent form. All subjects enrolled will be assigned a subject code, which consists 
of a consecutive subject number (2 digits). 
7.1.2 Inclusion criteria 
 
Male and female subjects will be eligible for participation in this study if they: 
• 
Are ≥18 years on the day of screening 
• 
Had undergone an allogeneic HSCT 11 to 13 months ago (study population) 
• 
Are clinical healthy without previous TBE vaccination (control group) 
• 
Have an understanding of the study, agree to its provisions, and give written informed 
consent prior to study entry 
• 
If female and capable of bearing children – have a negative urine pregnancy test 
result at study entry and agree to employ adequate birth control measures for the 
duration of the study 
7.1.3 Exclusion criteria 
 
Subjects will be excluded from participation in this study if they: 
• 
Have received a TBE vaccination following HSCT 

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• 
Suffer from extremely severe acute GVHD and therefore receive prednisone >0.5 
mg/kg bodyweight as part of a combination therapy or a three agent 
immunosuppressive treatment (because in these HSCT patients any type of 
vaccination has to be postponed until immunosuppression is reduced to a double 
combination or prednisone <0.5 mg/kg bodyweight) 
• 
Suffer from or have a history of previous TBE virus infection or vaccination, previous 
dengue virus infection or vaccination against yellow fever or Japanese encephalitis 
• 
Have any acute febrile illness in the 2 weeks prior to or at the time of enrolment 
• 
Have a history of severe allergic reactions or anaphylaxis after vaccination 
• 
If female, are pregnant or lactating. 
• 
If belonging to the healthy control group, are immunosuppressed (suffer from or have 
a history of immune mediated diseases, long-term use of corticosteroids, 
hemodialysis, chronic renal insufficiency, liver cirrhosis Child-Pugh class C, 
hematooncological malignant disease, solid organ transplant, HSCT) 
• 
Suffer a relapse of their underlying malignant disease 
7.1.4 Study duration 
 
The total study duration is estimated to be four years. For the individual study participant the 
study duration will be 44-56 weeks. 
7.1.5 Withdrawal and replacement of subjects 
 
Criteria for withdrawal 
 
Subjects may prematurely discontinue from the study at any time. Premature discontinuation 
from the study is to be understood when the subject did not undergo complete the last visit 
(study visit 8) and / or all pivotal assessments during the study. 
 
Subjects must be withdrawn under the following circumstances: 
 
• 
at their own request 
• 
if the investigator feels it would not be in the best interest of the subject to continue 
• 
if the subject violates conditions laid out in the consent form / information sheet or 
disregards instructions by the study personal 
 

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In all cases, the reason why subjects are withdrawn must be recorded in detail in the CRF 
and in the subject’s medical records. Should the study be discontinued prematurely, all study 
materials (complete, partially completed and empty CRFs) will be retained. 
 
Follow-up of patients withdrawn from the study 
 
In case of premature discontinuation after start of vaccination, no further investigations 
concerning the study will be performed. Furthermore, participants may request that from the 
time point of withdrawal no more data will be recorded and that all biological samples 
collected in the course of the study will be destroyed. 
 
Replacement policy 
 
If the week 8 (primary endpoint) is reached, drop-outs will be included in the sample size. If 
the participiant drops out before the week 8 is reached, patients will be replaced and the next 
free subject number will be allocated. 
 
7.1.6 Premature termination of the study 
 
The sponsor has the right to close this study at any time. The IEC and the competent 
regulatory authority must be informed within 15 days of early termination. 
The trial or single dose steps will be terminated prematurely in the following cases: 
• 
If adverse events occur which are so serious that the risk-benefit ratio is not 
acceptable. 
• 
If the number of dropouts is so high that proper completion of the trial cannot 
realistically be expected. 
8. METHODOLOGY 
 
8.1  Study medication 
 
Active agent and characteristics: Inactived TBE-virus adsorbat vaccine. One dose (0.5 ml) 
contains 2.4 µg of formaldehyde inactivated TBE-virus (strain Neudörfl) and 1 mg aluminium 
hydroxide as adjuvant. 
Trade name of the agent: FSME IMMUN 0.5 ml 
Manufacturer: PFIZER 
Drug supply: PFIZER 

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Storage Instructions: The vaccine has to be stored in a refrigerator at 2-8°C in the orginal 
package. 
Route of administration: intramuscular injection. 
 
8.1.1 Dosage and administration 
 
Initial dose: All subjects will receive one dose 0.5 ml TBE vaccine FSME IMMUN on day 0 
(study visit 1). 
Repeated dose: All subjects will receive one additional dose 0.5 ml TBE vaccine FSME 
IMMUN on week 4 (study visit 2) and between week 40-52 (study visit 6). 
Route of administration: TBE vaccine will be injected intramuscularly in the Musculus 
deltoideus. 
Duration: All subjects will receive 0.5 ml TBE vaccine three times within one year. 
 
8.1.2 Study-drug up- and down titration 
 
Not needed for the study. 
 
8.1.3 Study drug interruption or discontinuation 
 
The investigator must temporarily interrupt or permanently discontinue the study drug if 
continued administration of the study drug is believed to be contrary to the best interests of 
the patient. 
The interruption or premature discontinuation of study drug might be triggered by an AE, a 
diagnostic or therapeutic procedure, an abnormal assessment, or for administrative reasons, 
in particular withdrawal of the patient’s consent. 
The reason for study drug interruption or premature permanent discontinuation must be 
documented in the CRF. 
 
8.1.4 Study-drug delivery & drug storage conditions 
 
The drug will be ordered from the local pharmacy of the General Hospital of Vienna. After 
receipt the vaccine will be stored in the refrigerator at 2-8°C in the original package at the 
outpatient clinic of the Department of Internal Medicine I, Division of Infectious Diseases and 
Tropcial Medicine. The vaccine should not be frozen or stored at a temperature >8°C. 

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8.1.5 Study drug packaging and labeling 
 
Packaging and labeling of the TBE vaccine is done by the producer according to local legal 
requirements and GMP and shipped to the local pharmacy of the General Hospital of Vienna. 
8.1.6 IMP administration & handling 
 
Before application the vaccine has to reach room temperature. Furthermore, the suspension 
has to be thrilled and has to be injected intramuscularly in the musculus deltoideus 
immediately after the protecting cap is removed from the needle. Longer, unprotected resting 
can lead to unsterility and/or occlusion of the needle. All vaccines have to be documented 
with self-adhesive label in the vaccination card.  
8.1.7 Drug Accountability 
 
The application of the drug will be documented in the clinical records of the patient by using a 
self-adhesive label of the vaccine and in the CRF.  
8.1.8 
Procedures to assess subjects compliance 
 
Due to intramuscular injection by the doctor, the compliance of the patients has not to be 
additionally checked. 
8.1.9 
Concomitant medication 
 
Concomitant medication has to be documented in the CRF.  
 
8.2  Randomization 
 
Not needed for the study.  
8.3  Blinding 
 
Not needed for the study.  
8.4  Benefit and risk assessment 
 
After vaccination with the inactivated TBE virus K23, in particular following the administration 
of the complete primary immunization, it is highly probable that the majority of subjects 
participating in the phase II clinical study will be protected against TBE, one of the most 
severe infections of the central nervous system. Allogeneic HSCT recipients, particularly 
those with chronic GVHD, have a high risk for microbial infections including TBE infection. 
Therefore, primary vaccination of HSCT recipients is recommended in risk areas one year 

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after HSCT at the latest independently of previous TBE vaccination before HSCT. The used 
TBE-vaccine is registered in Austria for more than one decade. Its safety and efficacy have 
been already demonstrated in many clinical trials. Therefore, unexpected adverse events 
due to the vaccination are not assumed. However, the following side effects have to be 
considered: 
- very common (>1/10): local reactions at the site of injection, transient redness, and 
swelling. 
- common (>1/100;<1/10): systemic reactions including fever, fatigue, headache, muscular 
pain, arthralgia. These side-effects usually occur within 72 hours after the vaccination, abate 
within 1-2 days, but do not acquire any medical treatment. All adverse events will be listed in 
the study documents and have to evaluated by the investigator for causality to the 
vaccination. After venous puncture local reactions, a hematoma or phlebitis can occur. 
However, the planed venous punctures withdraw only minor volumes of blood and do not 
impair the patient’s health. 
Therefore, it can be speculated that this study does not exhibit an exceptional risk for the 
patients. 
 
8.5  Study procedures 
8.5.1 General rules for trial procedures 
 
• 
All study measures like blood sampling and measurements have to be documented 
with date (dd:mm:yyyy).  
• 
In case several study procedures are scheduled at the same time point, there is no 
specific sequence that should be followed. 
• 
The dates of all procedures should be according to the protocol. The time margins 
mentioned in the study flow chart are admissible. If for any reason, a study procedure 
is not performed within scheduled margins a protocol deviation should be noted, and 
the procedure should be performed as soon as possible or as adequate. 
• 
If it is necessary for organizational reasons, it is admissible to perform procedures 
which are scheduled for one visit at two different time points. Allowed time margins 
should thereby not be exceeded. 
 
8.5.2 Study visits 
8.5.2.1 Visit 0/ Screening investigation (Day -28) 
 

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The investigator will inform the subject about the procedures, risks and benefits oft he study. 
Fully informed, written consent must be obtained from each subject prior to any assessment 
being performed. It is important that the subject is allowed sufficient time to consider his/her 
participation in the study.  
The following assessments will be performed: 
• 
Inclusion and exclusion criteria. 
• 
Demographic data including sex, age, weight and height. 
• 
Medical history and information on planned hospitalization (including elective surgery) 
during the study for medical conditions existing prior to study entry. 
• 
Physical examination including body temperature measured axillary. 
• 
Pregnancy test in females capable of bearing children. 
• 
Blood draw of 10 ml blood for TBE antibody level detection. 
• 
Blood draw of 40 ml blood for cellular immunity. 
• 
Blood draw of 10 ml blood for immune status and quantitative immunoglobulin levels 
in HSCT patients only. 
Subjects who suffer from an acute illness with or without elevated body temperature (≥ 
37.5°C, measured axillary with two weeks prior to screening will not be vaccinated. In this 
case Visit 1 will take place separately at a later date, as long as the study is ongoing. 
8.5.2.2. 
Visit 1 /First Vaccination (Day 0) 
 
The following activities will be performed before vaccination only when Visit 0 and Visit 1 are 
not the same day 
• 
Review of inclusion and exclusion criteria 
• 
Medical history update 
• 
Physical examination including body temperature measured axillary 
• 
Pregnancy test in females capable of bearing children. 
 
Then the subject will receive the first vaccination. For vaccination the following procedures 
have to be considered: 
• 
The vaccine has to reach room temperature before administration of the vaccine 
• 
The vaccine has to be shaken vigorously for five to ten seconds 
• 
The injection site has to prepared according to standard clinical procedures 
• 
After removement of the protecting cap from the needle the 0.5 ml dose of the 
vaccine has to be administered immediately by intramuscular injection into the 
musculus deltoideus in the upper arm. 
 

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Following vaccination, the subject will be oserved for at least 30 minutes. Any infection site 
reactions and systemic adverse experiences will be recorded.  
8.5.2.3. 
Visit 2 /Second Vaccination (Day 28 ± 2 days) 
 
The following activities will be performed:  
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 1. All adverse experiences will be documented in the CRF 
• 
Physical examination including body temperature measured axillarily 
• 
Pregnancy test in females capable of bearing children. 
• 
Second vaccination: for details see above 
• 
Post vaccination observation period (30 minutes): for details see above 
8.5.2.4. 
Visit 3 /1. Evaluation of cellular immunity (Day 35, 7 ± 2 days after the 
second vaccination) 
 
The following activities will be performed:  
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 2. All adverse experiences will be documented in the CRF. 
• 
Blood draw of 40 ml for determination of cellular immunity 
8.5.2.5. 
Visit 4 /1. Evaluation of humoral immunity (Day 56, 28 ± 7 days after the 
second vaccination) 
 
The following activities will be performed:  
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 3. All adverse experiences will be documented in the CRF. 
• 
Blood draw of 10 ml for serum antibody determination (NT, ELISA) 
• 
Pregnancy test in females capable of bearing children. 
8.5.2.6. 
Visit 5 /1. Evaluation of immune status (Day 84 ± 14 days) 
 
The following activities will be performed in HSCT patients only: 
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 4. All adverse experiences will be documented in the CRF. 
• 
Blood draw of 10 ml for immune status and quantitative immunoglobulin levels 
• 
Pregnancy test in females capable of bearing children. 
8.5.2.7. 
Visit 6 /3. Vaccination and evaluation of immune status (Day 270-365) 
 

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The following activities will be performed:  
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 5. All adverse experiences will be documented in the CRF. 
• 
Physical examination including body temperature measured axillary. 
• 
Pregnancy test in females capable of bearing children. 
• 
Third vaccination: for details see Visit 1. 
• 
Post vaccination observation period (30 minutes): for details see Visit 1. 
• 
Blood draw of 10 ml for  immune status and quantitative immunoglobulin levels 
8.5.2.8. 
Visit 7 /2. Evaluation of cellular immunity (Day 277-372, 7 ± 2 days after 
the third vaccination) 
 
The following activities will be performed in HSCT patients only: 
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 6. All adverse experiences will be documented in the CRF. 
• 
Blood draw of 40 ml for cellular immunity 
8.5.2.9. 
Visit 8 /2. Evaluation of humoral immunity (Day 298-393, 28 ± 7 days after 
the third vaccination) 
 
The following activities will be performed in HSCT patients only: 
• 
The investigator will ask all subjects about any adverse experiences occurring since 
Visit 7. All adverse experiences will be documented in the CRF. 
• 
Blood draw of 10 ml for serum antibody determination (NT, ELISA) 
• 
Pregnancy test in females capable of bearing children. 
After this visit, the patients will have completed the study. 
 
8.5.3 Determination of the humoral and cellular immunity 
8.5.3.1  Determination of serum antibodies against vaccination antigens 
 
For the evaluation of antibody levels, both NT and enzyme linked immunosorbent assay 
(ELISA) will be performed [23]. Virus neutralization titers will be defined as the reciprocal of 
the serum dilution that gives a 90% reduction of virus growth compared to the control without 
antibody.  
Neutralizing antibodies have been shown to be protective [24] and are generally viewed as a 
surrogate parameter of protection against TBE [17]. Neutralization tests will be performed by 
PFIZER. Blood samples will be also screened for TBE antibodies by ELISA [23] and will be 
considered seropositive when the values are above the cut-off of the assay. 

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8.5.3.2  Determination of cellular immune response 
 
To investigate cellular immunity following TBE vaccination, lymphocyte proliferation and 
cytokine detection assays will be used. Peripheral blood mononuclear cells (PBMCs) will be 
isolated from heparinized venous blood by ficoll-diatrizoate centrifugation at 3 points in time 
as specified above (study visit 1, 3 and 7) and will be cryopreserved and stored in liquid 
nitrogen for later use. Lymphocyte proliferation will be analysed by [3H]thymidine 
incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling. PBMCs 
from defined time points for each subject will be thawed [25] and assayed together. As 
antigen to stimulate PBMCs we will use the whole aluminium hydroxide-free and human 
albuminfree TBE antigen at 4.8 µg/ml provided by PFIZER. Phytohemagglutinin (PHA) will be 
used for nonspecifically mitogenic stimulation of PBMCs at either 1 or 5 µg/ml. PBMCs will 
be cultured in triplicate (105 PBMC/well) with or without various stimuli at 37°C in a CO2 
incubator for 6 days before harvesting. For the [3H]thymidine incorporation assay, PBMCs 
will be then pulsed with radioactive 3H (tritiated) thymidine for sixteen hours. The amount of 
radioactivity incorporated into DNA in each well will be measured in a scintillation counter 
and is proportional to the number of proliferating cells, which in turn is a function of the 
number of lymphocytes that were stimulated by a given antigen to enter the proliferative 
response. Results will be expressed as Δcpm (calculated as mean counts per minute of 
stimulated cell cultures minus mean counts per minute of medium control cell cultures) and 
as the stimulation index (SI), calculated as the ratio of the mean counts per minute of 
stimulated cell cultures to the mean counts per minute of medium control cell cultures [26]. 
For the CFSE proliferation assay, PBMCs will be CFSE-loaded (and fluorescein 
isothiocyanate-labeled) and cultured with medium alone (negative control), or PHA, or the 
whole aluminium hydroxide-free and human albumin-free TBE antigen at 4.8 µg/ml. After 6 
days of incubation at 37°C and 5%CO2, collected cells will be stained with anti-CD3 
monoclonal antibody (allophycocyanin), anti-CD8 (peridinin chlorophyll protein), and anti-
CD4 (phycoerythrin). A total of 100,000 events in a lymphocyte gate will be analyzed by flow 
cytometry. Percentages of CD3+, CD8+, and CD4+ CFSE proliferating cells will be evaluated 
simultaneously by gating on CD3+ T cells and measuring sequentially proliferating CD8+ and 
CD4+ subsets within the CD3+ subsets. Net percentages of CFSE will be calculated by 
subtracting the negative control values as described previously [27]. Furthermore, cytokines 
will be measured in the supernatant (IL-2, IFN-gamma, IL-6, IL-10, TNFalpha) after in vitro 
stimulation with and without TBE virus antigen or PHA for 48 hours using ELISA. 

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8.5.3.3 
 Determination of immune reconstitution in HSCT recipients 
 
The status of immune reconstitution in HSCT recipients prior to and after vaccination will be 
assessed by immunofluorescence staining of peripheral blood (PB) T and B lymphocytes and 
flow cytometric analyses. Therefore, PB cells will be stained with monoclonal antibodies and 
analyzed by FACS for the following cell populations: T lymphocytes (CD3+), B lymphocytes 
(CD19+), T helper cells (CD3+CD4+), T suppressor cells (CD3+CD8+), natural killer cells 
(CD3-CD16&56+) and cytotoxic cells (CD3+CD16&56+). 
In addition, serum immunoglobulin levels will be analysed. Immune reconstitution and serum 
immunoglobulin levels will be determined in all HSCT patients prior to the first and third 
vaccination and 12 weeks after the first vaccination (study visits 1, 5 and 6). 
 
8.5.4 Study endpoints 
8.5.4.1 Primary study endpoint 
 
To assess the humoral immunogenicity of the TBE vaccination, the outcome of NT will be 
analysed four weeks after the second vaccination. Therefore, the number of subjects with NT 
titers against TBE virus >10, assumed to be the threshold for antibody-mediated protection, 
will be evaluated. 
8.5.4.2 Secondary study end points 
 
The secondary end points of this study are: 
• 
Antibody concentrations of TBE ELISA (validated in-house test, Department of 
Virology, Medical University of Vienna) before and four weeks after the second and 
third vaccination. 
o Increase of antibody response. 
o Number of subjects with seroconversion defined when patients reach antibody 
levels above the cut-off of TBE ELISA. 
• 
Antibody concentrations of NT titers four weeks after the third vaccination compared 
to four weeks after the second vaccination.  
o Increase of antibody response. 
o Number of subjects with NT titers against TBE virus >10. 
• 
Evaluation of cellular immunity prior to and one week after the second and third 
vaccination. 
o Lymphoproliferative response after vaccination induced by TBE antigen. 
o Increase in cytokine levels induced by TBE antigen 

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• 
Evaluation of immune reconstitution and quantitative immunoglobulin levels before 
first and third vaccination and 12 weeks after first vaccination in HSCT recipients only. 
 
9. SAFETY DEFINITIONS AND REPORTING REQUIREMENTS 
9.1  Adverse events (AEs) 
9.1.1 Summary of known and potential risks of the study drug 
 
The following side effects have to be considered: 
- very common (>1/10): local reactions at the site of injection, transient redness, and 
swelling. 
- common (>1/100;<1/10): systemic reactions including fever, fatigue, headache, muscular 
pain, arthralgia. These side-effects usually occur within 72 hours after the vaccination, abate 
within 1-2 days, but do not acquire any medical treatment. All adverse events will be listed in 
the study documents and have to evaluated by the investigator for causality to the 
vaccination. After venous puncture local reactions, a hematoma or phlebitis can occur. 
However, the planed venous punctures withdraw only minor volumes of blood and do not 
impair the patient’s health. 
 
9.1.2 Definition of adverse events 
 
An AE is any untoward adverse change from the subject's baseline condition, i.e., any 
unfavorable and unintended sign including an abnormal laboratory finding, symptom or 
disease which is considered to be clinically relevant by the physician that occurs during the 
course of the study, whether or not considered related to the study drug. 
Adverse events include: 
• 
Exacerbation of a pre-existing disease. 
• 
Increase in frequency or intensity of a pre-existing episodic disease or medical 
condition. 
• 
Disease or medical condition detected or diagnosed after study drug administration 
even though it may have been present prior to the start of the study. 
• 
Continuous persistent disease or symptoms present at baseline that worsen following 
the start of the study. 
• 
Lack of efficacy in the acute treatment of a life-threatening disease. 
• 
Events considered by the investigator to be related to study-mandated procedures. 

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• 
Abnormal assessments, e.g., ECG and physical examination findings, must be 
reported as AEs if they represent a clinically significant finding that was not present at 
baseline or worsened during the course of the study. 
• 
Laboratory test abnormalities must be reported as AEs if they represent a clinically 
significant finding, symptomatic or not, which was not present at baseline or 
worsened during the course of the study or led to dose reduction, interruption or 
permanent discontinuation of study drug. 
Adverse events do not include:  
• 
Pre-planned interventions or occurrence of endpoints specified in the study protocol 
are not considered AE´s, if not defined otherwise (eg.as a result of overdose) 
• 
Medical or surgical procedure, e.g., surgery, endoscopy, tooth extraction, transfusion. 
However, the event leading to the procedure is an AE. If this event is serious, the 
procedure must be described in the SAE narrative. 
• 
Pre-existing disease or medical condition that does not worsen. 
• 
Situations in which an adverse change did not occur, e.g., hospitalizations for 
cosmetic elective surgery or for social and/or convenience reasons. 
• 
Overdose of either study drug or concomitant medication without any signs or 
symptoms. However, overdose must be mentioned in the Study Drug Log. 
 
9.2  Serious Adverse Events (SAEs) 
 
A Serious Adverse Event (SAE) is defined by the International Conference on Harmonization 
(ICH) guidelines and WHO GCP guidelines as any AE fulfilling at least one of the following 
criteria: 
• 
Results in deaths. 
• 
Life-threatening – defined as an event in which the subject was, in the judgment of 
the investigator, at risk of death at the time of the event; it does not refer to an event 
that hypothetically might have caused death had it been more severe. 
• 
Requiring subject's hospitalization or prolongation of existing hospitalization – 
inpatient hospitalization refers to any inpatient admission, regardless of length of stay. 
• 
Resulting in persistent or significant disability or incapacity (i.e., a substantial 
disruption of a person’s ability to conduct normal life functions). 
• 
Congenital anomaly or birth defect. 
• 
Is medically significant or requires intervention to prevent at least one of the 
outcomes listed above. 

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Life-threatening refers to an event in which the subject was at risk of death at the time of the 
event. It does not refer to an event that hypothetically might have caused death if it were 
more severe. 
Important medical events that may not immediately result in death, be life-threatening, or 
require hospitalization may be considered as SAEs when, based upon appropriate medical 
judgment, they may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed in the definitions above. 
 
9.2.1 Hospitalization – Prolongation of existing hospitalization 
 
Hospitalization is defined as an overnight stay in a hospital unit and/or emergency room. 
An additional overnight stay defines a prolongation of existing hospitalization. 
The following is not considered an SAE and should be reported as an AE only: 
• 
Treatment on an emergency or outsubject basis for an event not fulfilling the 
definition of seriousness given above and not resulting in hospitalization. 
The following reasons for hospitalizations are not considered AEs, and therefore not SAEs: 
• 
Hospitalizations for cosmetic elective surgery, social and/or convenience reasons. 
• 
Standard monitoring of a pre-existing disease or medical condition that did not 
worsen, e.g., hospitalization for coronary angiography in a subject with stable angina 
pectoris. 
• 
Elective treatment of a pre-existing disease or medical condition that did not worsen, 
e.g., hospitalization for chemotherapy for cancer, elective hip replacement for 
arthritis. 
 
9.2.2 SAEs related to study-mandated procedures 
 
Such SAEs are defined as SAEs that appear to have a reasonable possibility of causal 
relationship (i.e., a relationship cannot be ruled out) to study-mandated procedures 
(excluding administration of study drug) such as discontinuation of subject's previous 
treatment during a washout period, or complication of a mandated invasive procedure (e.g., 
blood sampling, heart catheterization), or car accident on the way to the hospital for a study 
visit, etc. 
 
9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
 

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SUSARs are all serious adverse reactions with suspect causal relationship to the study drug 
that is unexpected (not previously described in the SmPC - Summary of Product 
Characteristics or Investigator’s brochure) and serious.  
 
9.2.4 Pregnancy 
 
Any pregnancy that occurs during study participation must be reported to the 
investigator/sponsor. To ensure subject safety, each pregnancy must be reported to the 
investigator/sponsor immediately. The pregnancy must be followed up to determine outcome 
(including premature termination) and status of mother and child.  Pregnancy complications 
and elective terminations for medical reasons must be reported as an AE or SAE.  
Spontaneous abortions must be reported as an SAE. 
Any SAE occurring in association with a pregnancy brought to the investigator’s attention 
after the subject has completed the study and considered by the investigator as possibly 
related to the investigational product, must be promptly reported to the principal 
investigator/sponsor. 
In addition, the investigator must attempt to collect pregnancy information on any female 
partners of male study subjects who become pregnant while the subject is enrolled in the 
study. Pregnancy information must be reported to the investigator/sponsor as described 
above. 
 
9.3  Severity of adverse events 
 
The severity of clinical AEs is graded on a three-point scale: mild, moderate, severe, and 
reported on specific AE pages of the CRF. 
If the severity of an AE worsens during study drug administration, only the worst intensity 
should be reported on the AE page. If the AE lessens in intensity, no change in the severity 
is required.  
 
Mild 
Event may be noticeable to subject; does not influence daily activities; the AE resolves 
spontaneously or may require minimal therapeutic intervention;  
 
Moderate 
Event may make subject uncomfortable; performance of daily activities may be influenced; 
intervention may be needed; the AE produces no sequelae. 

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Severe 
Event may cause noticeable discomfort; usually interferes with daily activities; subject may 
not be able to continue in the study; the AE produces sequelae, which require prolonged 
therapeutic intervention. 
A mild, moderate or severe AE may or may not be serious. These terms are used to describe 
the intensity of a specific event (as in mild, moderate, or severe myocardial infarction). 
However, a severe event may be of relatively minor medical significance (such as severe 
headache) and is not necessarily serious. For example, nausea lasting several hours may be 
rated as severe, but may not be clinically serious. Fever of 39°C that is not considered 
severe may become serious if it prolongs hospital discharge by a day. Seriousness rather 
than severity serves as a guide for defining regulatory reporting obligations. 
 
9.4  Relationship to study drug 
 
For all AEs, the investigator will assess the causal relationship between the study drug and 
the AE using his/her clinical expertise and judgment according to the following algorithm that 
best fits the circumstances of the AE: 
Unrelated 
• 
May or may not follow a reasonable temporal sequence from administration of the 
study product 
• 
Is biologically implausible and does not follow known response pattern to the suspect 
study drug (if response pattern is previously known). 
• 
Can be explained by the known characteristics of the subject’s clinical state or other 
modes of therapy administered to the subject. 
• 
Unlikely 
• 
May or may not follow a reasonable temporal sequence from administration of the 
study product 
• 
Is biologically not very plausible  
• 
May be explained by the known characteristics of the subject’s clinical state or other 
modes of therapy administered to the subject. 
 
Possible related 
• 
Follows a reasonable temporal sequence form administration of the study drug. 

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• 
May follow a known response pattern to the study drug (if response pattern is 
previously known). 
• 
Could not be reasonably explained by the known characteristics of the subject’s 
clinical state or other modes of therapy administered to the subject, if applicable. 
• 
Probable 
• 
Follows a reasonable temporal sequence form administration of the study drug. 
• 
Follows a known response pattern to the study drug (if response pattern is previously 
known). 
• 
other causes for the event are unlikely 
 
Definitely related 
• 
Follows a reasonable temporal sequence form administration of the study drug. 
• 
Follows a known response pattern to the study drug (if response pattern is previously 
known). 
• 
No other reasonable cause is present. 
 
9.5  Reporting procedures 
 
A special section is designated to adverse events in the case report form. The following 
details must thereby be entered: 
• 
Type of adverse event 
• 
Start (date and time) 
• 
End (date and time) 
• 
Severity (mild, moderate, severe) 
• 
Serious (no / yes) 
• 
Unexpected (no / yes)  
• 
Outcome (resolved, ongoing, ongoing – improved, ongoing – worsening) 
• 
Relation to study drug (unrelated, possibly related, definitely related) 
Adverse events are to be documented in the case report form in accordance with the above 
mentioned criteria. 
 
9.5.1 Reporting procedures for SAEs  
 

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In the event of serious, the investigator has to use all supportive measures for best patient 
treatment. A written report is also to be prepared and made available to the clinical 
investigator immediately. The following details should at least be available: 
• 
Patient initials and number 
• 
Patient: date of birth, sex, ethical origin 
• 
The suspected investigational medical product (IMP) 
• 
The adverse event assessed as serious 
• 
Short description of the event and outcome 
If applicable, the initial report should be followed by the Follow up report, indicating the 
outcome of the SAE. 
 
9.5.2 Reporting procedures for SUSARs 
 
It must be remembered that the regulatory authorities, and in case of SUSARs which could 
possibly concern the safety of the study participants, also the Institutional Review Board / 
Independent Ethics Committee (IRB / IEC) are to be informed. Such reports shall be made 
by the study management and the following details should be at least available: 
• 
Patient initials and number 
• 
Patient: date of birth, sex, ethical origin 
• 
Name of investigator and investigating site 
• 
Period of administration 
• 
The suspected investigational medical product (IMP) 
• 
The adverse event assessed as serious and unexpected, and for which there is a 
reasonable suspected causal relationship to the IMP 
• 
Concomitant disease and medication 
• 
Short description of the event: 
• 
Description 
• 
Onset and if applicable, end 
• 
Therapeutic intervention 
• 
Causal relationship 
• 
Hospitalization of prolongation of hospitalization 
• 
Death, life-threatening, persistent or significant disability or incapacity 
Electronic reporting should be the expected method for reporting of SUSARs to the 
competent authority. In that case, the format and content as defined by Guidance (28) should 

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be adhered to. The latest version of MedDRA should be applied. Lower level terms (LLT) 
should be used. 
 
9.5.3 Annual Safety Report 
 
The Annual Safety Report will be provided by the principal investigator at least once a year. 
This report will also be presented annually to the Independent Ethics (IEC) and to the 
competent authorities by the sponsor. 
 
10. 
FOLLOW-UP 
 
No follow-up of the patient is required for this study. 
 
11. 
STATISTICAL ANALYSIS 
 
Statistical analysis will be planed and performed by a statistician of the Institute for Medical 
Statistics, Medical University of Vienna, Dr. Alexandra Graf. 
11.1 
Sample size considerations 
 
For our phase II pilot study a calculation of the sample size was performed using nQuery 6.1. 
The primary endpoint is the outcome of the NT against 4 weeks after the second vaccination 
(positive test: Yes/No). Because of deviation of the antibody concentrations from normal 
distribution (NT level <=10 not detectable indicating a negative test), the outcome is reduced 
to a binary but clinically more relevant outcome (seroconversion rate: NT levels >10 indicates 
a positive test). Previously, in a study with heart transplant recipients a seroconversion rate 
of 35% was detected compared to 100% in the healthy control group [21]. In our study we 
expect a seroconversion rate of at least 90% in the healthy control group. In the HSCT 
patients we expect a seroconversion not exceeding 50%. The calculation of the sample size 
was performed using the following assumption: Significance level alpha 0.05, Power 0.8, 
ratio of HSCT patients/healthy volunteers: 1/1. With a total number of 46 participants (23 
HSCT patients and 23 healthy volunteers) the assumed difference in the seroconversion rate 
between groups can be detected (using a two-sided Fisher’s Exact Test) with 80% Power. 
We added 10% to adjust for potential loss-to-follow-up which results in 26 HSCT patients and 
26 healthy volunteers. 

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11.2 
Relevant protocol deviations 
 
All protocol deviations will be listed in the study report. Major deviations regarding to subjects 
safety will lead to withdrawal. 
 
11.3 
Endpoints analysis 
 
The primary endpoint (outcome of NT 4 weeks after the second vaccination) will be analysed 
using Fisher’s Exact Test. For the seroconversion rates, 95% confidence intervals will be 
calculated. All subjects with available antibody responses fulfilling the eligibility criteria will be 
included in the analysis 
The secondary endpoints (NT 4 weeks after the third vaccination, ELISA prior to the first 
vaccination and 4 weeks after the second and third vaccination, cellular immunity, 
immunoglobulin levels and immune reconstitution) will be analysed using descriptive 
statistics. 
 
11.4 
Missing, unused and spurious data 
 
Only subjects for whom data are available will be included in the statistical analysis. Missing 
values will neither be replaced nor estimated.  
11.5 
Interim analysis 
 
No interim analysis will be performed.  
11.6 
Software program(s) 
 
Sample size calculation was performed using nQuery 6.1. 
Statistical analysis will be prepared using SPSS Statistics (Version 17.0 or higher). 
12. 
DOCUMENTATION AND DATA MANAGEMENT 
 
12.1 
Documentation of study results 
 
A subject screening and enrollment Log will be completed for all eligible or non-eligible 
subjects with the reasons for exclusion. 
 

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12.1.1 Case report form (CRF) 
 
For each subject enrolled, regardless of study drug initiation, a Paper-CRF must be 
completed and signed by the investigator or a designated sub-investigator. This also applies 
to those subjects who fail to complete the study. If a subject withdraws from the study, the 
reason must be noted on the CRF. Case report forms are to be completed on an ongoing 
basis. 
CRF entries and corrections will only be performed by study site staff, authorized by the 
investigator.  
In the “Paper-CRF” all forms should be completed and must be legible. Errors should be 
crossed out but not obliterated, the correction inserted, and the change initialed and dated by 
the investigator, co-investigator or study nurse. The original CRFs are passed first by Dr. 
Forstner and then the entries will be checked by the monitor Dr. Ramharter and any errors or 
inconsistencies will be checked immediately.  
The monitor will collect original completed and signed CRFs at the end of the study.  
 
12.1.2 Data Collection 
 
Data collected at all visits are entered into an interactive form. The CRFs will be source 
documents verified following guidelines established before study onset as detailed in the 
Monitoring Plan. Maintenance of the study database will be performed by Dr. Forstner. 
 
12.2 
Safekeeping 
 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified (according to 
ICH-GCP “essential documents”). These documents will be classified into two different 
categories: investigator's file, and subject clinical source documents. 
The investigator's file will contain the protocol/amendments, EudraCT forms, CRFs (eCRF 
printout), standard operation procedures (SOPs), data clarification and query forms, EC/IRB 
and Health Authority approval with correspondence, informed consent, drug records, staff 
curriculum vitae and authorization forms, screening and enrollment logs, and other 
appropriate documents/correspondence as per ICH/Good Clinical Practice (GCP) and local 
regulations. 
Subject clinical source documents include, but are not limited to subject hospital/clinic 
records, physician’s and nurse’s notes, appointment book, original laboratory reports, ECG, 

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X-ray, pathology and special assessment reports, consultant letters, etc. 
 
These two categories of documents must be kept on file by the investigator for as long as 
needed to comply with national and international regulations (in Austria 15 years after 
discontinuing clinical development or after the last marketing approval). If source documents 
are not durable as long as needed they must be preserved as a copy. No study document 
should be destroyed without prior written approval from the Department of Internal Medicine 
I. 
When source documents are required for the continued care of the subject, appropriate 
copies should be made for storing outside of the site. 
 
12.3 
Quality Control and Quality Assurance 
 
The following quality control and quality assurance measures will be taken to ensure the 
adherence to GCP and applicable regulatory requirements as well as the accuracy and 
integrity of data obtained from the study: 
• 
Periodic visits will be performed by the monitor to assure integrity and accuracy of 
study data. Discrepancies will be addressed and appropriate corrective action will be 
implemented.  
• 
Training for study personnel as well as for monitors will be provided on the correct 
handling and use of the CRF. Investigators will also be trained on GCP and Good 
Documentation Practice (GDP) relevant issues. 
 
12.3.1 Periodic Monitoring 
 
The designated monitor will contact and visit the investigator regularly and will be allowed to 
have access to all source documents needed to verify the entries in the CRFs and other 
protocol-related documents provided that subject confidentiality is maintained in agreement 
with local regulations. It will be the monitor's responsibility to inspect the CRFs at regular 
intervals according to the monitoring plan throughout the study, to verify the adherence to the 
protocol and the completeness, consistency and accuracy of the data being entered on them. 
The monitoring standards require full verification for the presence of informed consent, 
adherence to the inclusion/exclusion criteria, documentation of SAEs and the recording of 
the main efficacy, safety, and tolerability endpoints. 
The monitor will be working according to SOPs and will provide a monitoring report after 
each visit for the Sponsor. Depending on the quality of the data, additional monitoring visits 

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may be necessary according to the sponsor’s discretion. The investigator will resolve 
discrepancies of data. 
 
Monitoring will be performed by Dr. Ramharter. Three visits are planned, an initiation visit, 
one routine visit after the first year and a close out visit after the last patient has finished the 
study or data base lock. 100% of source data will be checked by the monitor (which means 
100% Source Data Verification). 
 
12.3.2 Audit and Inspections 
 
Upon request, the investigator will make all study-related source data and records available 
to a qualified quality assurance auditor mandated by the sponsor or to competent authority 
inspectors. The main purposes of an audit or inspection are to confirm that the rights and 
welfare of the subjects have been adequately protected, and that all data relevant for 
assessment of safety and efficacy of the investigational product have appropriately been 
reported to the sponsor. 
 
12.4 
Reporting and Publication  
 
12.4.1 Publication of study results  
 
The findings of this study will be published by the investigator) in a scientific journal and will 
be presented at scientific meetings. The manuscript will be circulated to all co-investigators 
before submission. Confidentiality of subjects in reports/publications will be guaranteed.  
 
13. 
ETHICAL AND LEGAL ASPECTS 
13.1 
Informed consent of subjects 
 
Following comprehensive instruction regarding the nature, significance, impact and risks of 
this clinical trial, the patient must give written consent to participation in the study. 
During the instruction the trial participants are to be made aware of the fact that they can 
withdraw their consent – without giving reasons – at any time without their further medical 
care being influenced in any way. 

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In addition to the comprehensive instructions given to the trial participants by the investigator, 
the trial participants also receive a written patient information sheet in comprehensible 
language, explaining the nature and purpose of the study and its progress. 
The patients must agree to the possibility of study-related data being passed on to relevant 
authorities. 
The patients must be informed in detail of their obligations in relation to the trial participants 
insurance in order not to jeopardize insurance cover. 
 
13.2 
Acknowledgement / approval of the study 
 
The investigator (or a designated CRO) will submit this protocol and any related document 
provided to the subject (such as subject information used to obtain informed consent) to an 
Ethics Committee (EC) or Institutional Review Board (IRB). Approval from the committee 
must be obtained before starting the study.  
The clinical trial shall be performed in full compliance with the legal regulations according to 
the Drug Law (AMG - Arzneimittelgesetz) of the Republic of Austria. 
An application must also be submitted to the Austrian Competent Authorities (Bundesamt für 
Sicherheit im Gesundheitswesen (BASG) represented by the Agency for Health and Food 
Safety (AGES PharmMed) and registered to the European Clinical Trial Database (EudraCT) 
using the required forms. The timelines for (silent) approval set by national law must be 
followed before starting the study. 
 
13.2.1 Changes in the Conduct of the Study 
 
 
Protocol amendments 
Proposed amendments must be submitted to the appropriate CA and ECs. Substantial 
amendments may be implemented only after CA/EC approval has been obtained. 
Amendments that are intended to eliminate an apparent immediate hazard to subjects may 
be implemented prior to receiving CA/EC approval. However, in this case, approval must be 
obtained as soon as possible after implementation. 
 
Study Termination 
If the sponsor or the investigator decides to terminate the study before it is completed, they 
will notify each other in writing stating the reasons of early termination. In terminating the 
study, the sponsor and the investigator will ensure the adequate consideration is given to the 
protection of the subject interests. The investigator, sponsor or (designated CRO on behalf of 
the sponsor) will notify the relevant CA and EC. 

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Clinical Study Report (CSR) 
Within one year after the final completion of the study, a full CSR will be prepared by the 
sponsor and submitted to the EC and the competent authority. 
The Investigator will be asked to review and sign the final study report. 
 
13.3 
Insurance 
 
During their participation in the clinical trial the patients will be insured as defined by legal 
requirements. The investigator of the clinical trial will receive a copy of the insurance 
conditions of the 'patients insurance'. The sponsor is providing insurance in order to 
indemnify (legal and financial coverage) the investigator/center against claims arising from 
the study, except for claims that arise from malpractice and/or negligence. The compensation 
of the subject in the event of study-related injuries will comply with the applicable regulations. 
Details on the existing patients insurance are given in the patient information sheet. 
 
13.4 
Confidentiality  
 
The information contained in this document, especially unpublished data, is the property of 
the project leader. It is therefore provided to you in confidence as an investigator, potential 
investigator, or consultant, for review by you, your staff, and an Ethics Committee or 
Institutional Review Board. It is understood that this information will not be disclosed to 
others without written authorization of the project leader. 
 
13.5 
Ethics and Good Clinical Practice (GCP) 
 
The investigator will ensure that this study is conducted in full conformance with the 
principles of the "Declaration of Helsinki" (as amended at the 56th WMA General Assembly, 
Tokyo, Japan, 2008) and with the laws and regulations of the country in which the clinical 
research is conducted. 
The investigator of the clinical trial shall guarantee that only appropriately trained personnel 
will be involved in the study. All studies must follow the ICH GCP Guidelines (June 1996) 
and, if applicable, the Code of Federal Regulations (USA). In other countries in which GCP 
Guidelines exist, the investigators will strictly ensure adherence to the stated provisions. 
Therefore this study follows the EU Directive embedded in the Austrian drug act. 
 
 
 

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14. 
APPENDICES 
 
Appendix 1. Informed Consent Form (Version 5.0; Date 30.01.2014) 
Appendix 2. Summary of medicinal products characteristics of FSME-IMMUN 0,5 ml 
 
 

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REFERENCES 
 
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