A Phase II Randomized, Placebo-Controlled, Multicenter Trial to Evaluate the Protective Function of a CMV-MVA Triplex Vaccine in Recipients of an Allogeneic Hematopoietic Stem Cell Transplant
Phase: PHASE2
Conditions: Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive, Acute Lymphoblastic Leukemia in Remission, Acute Myeloid Leukemia in Remission, Chronic Lymphocytic Leukemia, Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive, Cytomegaloviral Infection, Hodgkin Lymphoma, Lymphadenopathy, Lymphoblastic Lymphoma, Myelodysplastic Syndrome, Myelofibrosis, Myeloproliferative Neoplasm, Non-Hodgkin Lymphoma
Sponsor: City of Hope Medical Center

 
IRB #14295 
 
         12/15/2022    
Packet:  22  
CITY OF HOPE MEDICAL CENTER 
1500 E. DUARTE ROAD 
DUARTE, CA 91010 
 
Department of Hematology and Hematopoietic Cell Transplantation      
                          
                      
TITLE: A PHASE II RANDOMIZED,PLACEBO-CONTROLLED, MULTICENTER TRIAL TO EVALUATE 
PROTECTIVE FUNCTION OF CMV-MVA TRIPLEX VACCINE IN RECIPIENTS OF AN ALLOGENEIC 
HEMATOPOIETIC STEM CELL TRANSPLANT 
 
 
CITY OF HOPE PROTOCOL VERSION: 
IRB #14295 
Protocol Date: 12/15/2022 
 
DATE(S)/ OF AMENDMENT(S)/REVISION(S): 
 
 
COH Initial Approval  
Protocol Dated 07/30/15 
Version 00 
COH Amendment 01  
Protocol Dated 09/01/15 
Version 01  
COH Amendment 02  
Protocol Dated 11/03/15 
Version 02  
COH Amendment 03  
Protocol Dated 11/16/15 
Version 03  
COH Amendment 04  
Protocol Dated 12/30/15 
Version 04  
COH Amendment 05  
Protocol Dated 01/26/16 
Version 05  
COH Amendment 06  
TP only Dated  02/02/16 
Version 06  
COH Amendment 07  
Protocol Dated 03/07/16 
Version 07  
COH Amendment 08  
Protocol Dated 04/18/16 
Version 08  
COH Amendment 09  
Protocol Dated 05/10/16 
Version 09  
COH Amendment 10  
Protocol Dated 06/09/16 
Version 10  
COH Amendment 11  
Protocol Dated 07/11/16 
Version 11  
COH Amendment 12  
Protocol Dated 07/20/16 
Version 12  
COH Amendment 13              Protocol Dated 12/20/16 
                                                      Version 13 
COH Amendment 14              Protocol Dated 01/19/17 
                                                      Version 14 
COH Amendment 15              Protocol Dated 04/03/17 
                                                      Version 15 
COH Amendment 16              Protocol Dated 05/12/17 
                                                      Version 16 
COH Amendment 17              Protocol Dated 07/18/17 
                                                      Version 17 
COH Amendment 18              Protocol Dated 10/03/17 
                                                      Version 18 
COH Amendment 19  
Title Page Dated 02/25/19  
 
 
 
 
      Version 19  
COH Amendment 20  
Title Page Dated 02/26/20 
 
 
 
 
       Version 20 
COH Amendment 21  
Protocol Dated 10/03/17 TP  
 
 
 
       Packet 21 
COH Amendment 22  
Protocol Dated 12/15/2022   
 
 
 
       Packet 22 
 
DISEASE SITE:                                  Hematopoietic System 
    
   
  SPONSOR/IND NUMBER: 
             
City of Hope / IND# 15792 
 
TYPE : 
 
 
 
Phase II, Therapeutic  
 
PRINCIPAL INVESTIGATORS: 
Ryotaro Nakamura, M.D.   
 
 
 
COLLABORATING INVESTIGATORS: 
Ibrahim Aldoss, M.D., Don J. Diamond Ph.D.; Corinna La Rosa, 
Ph.D  

 
IRB #14295 
 
         12/15/2022    
Packet:  22  
 
 
 
 
PARTICIPATING CLINICIANS: 
Haris Ali, M.D., Ibrahim Aldoss, M.D., Monzr Al Malki, M.D., 
Elizabeth Budde, M.D., Ji-Lian Cai, M.D., Thai Cao, M.D., Len Farol, 
M.D., Stephen Forman, M.D., Alex Herrera, M.D., Myo Htut, M.D., 
Amrita Krishnan, M.D., Guido Marcucci, M.D., Matthew Mei, M.D., 
Liana Nikolaenko, M.D., Leslie Popplewell, M.D., Vinod Pullarkat, 
M.D., Firoozeh Sahebi, M.D., Amandeep Salhotra, Karamjeet 
Sandhu, M.D., M.D.,Tanya Siddiqi, M.D., Eileen Smith, M.D., 
Ricardo Spielberger, M.D., Anthony Stein, M.D., Sean (Shirong) 
Wang, M.D., Michael Rosenzweig, M.D, Ahmed Aribi, M.D, Nitya 
Nathwani, M.D., John Zaia, M.D., Jasmine Zain, M.D.  
 
 
STUDY STATISTICIAN:         
Dongyun Yang, Ph.D. 
 
 
PARTICIPATING SITES:  
City of Hope, Duarte, CA (Lead Institution),                                                             
                                               Dana-Farber Cancer Institute, Boston, MD Anderson Cancer Center, TX 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
CITY OF HOPE NATIONAL MEDICAL CENTER 
1500 E. DUARTE ROAD, DUARTE, CA 91010 
DEPARTMENT OF HEMATOLOGIC ONCOLOGY AND HEMATOPOIETIC CELL TRANSPLANTATION 
TITLE:  A PHASE II RANDOMIZED, PLACEBO-CONTROLLED, MULTICENTER TRIAL TO EVALUATE THE 
PROTECTIVE FUNCTION OF A CMV-MVA TRIPLEX VACCINE IN RECIPIENTS OF AN ALLOGENEIC 
HEMATOPOIETIC STEM CELL TRANSPLANT  
 
CITY OF HOPE PROTOCOL NUMBER: 
#14295 
VERSION: 19 
SPONSOR/IND NUMBER: 
City of Hope / IND #15792 
 
PRINCIPAL INVESTIGATORS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ryotaro Nakamura M.D. (Study and Coordinating PI) 
Department of Hematologic Oncology and Hematopoietic Cell 
Transplantation, City of Hope National Medical Center 
1500 E. Duarte Road, Duarte, CA 
Phone: 626-256-4673 
Fax: 626-301-8116 
e-mail: RNakamura@coh.org 
 
Ibrahim Aldoss, M.D. (Site PI) 
Department of Hematologic Oncology and Hematopoietic Cell 
Transplantation, City of Hope National Medical Center 
1500 E. Duarte Road, Duarte, CA 
Phone: 626-256-4673 
Fax: 626-301-8116 
e-mail: IAldoss@coh.org 
 
 
Lindsey Baden, M.D. (site PI) 
Primary Address: Brigham and Women’s Hospital, Infectious Disease 
Division, 75 Francis Street, PBB-4th Floor 
Boston, MA 02115 
Phone: 617-632-60664 
Fax:  617-732-6829 
e-mail: LBADEN@PARTNERS.ORG 
 
Ella Ariza-Heredia, M.D. (site PI) 
MD Anderson Cancer Center, Infectious Diseases, Infection Control 
1515 Holcombe Blvd, Houston, TX 77030 
Phone: 713-792-3627 
Fax:713-745-6839 
e-mail: Ariza@mdanderson.org 
 
 
Additional Location Where Clinical Investigation will be Conducted: 
Dana-Farber Cancer Institute, 
Blood and Marrow Transplant Program 
450 Brookline Ave, Boston, MA 02115  

 
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COLLABORATING INVESTIGATORS 
 
Don J. Diamond, Ph.D. 
Department of Experimental Therapeutics 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 
Phone: 626-256-4673 
Fax: 626-301-8981 
e-mail: DDiamond@coh.org 
 
Corinna La Rosa, Ph.D. 
Department of Experimental Therapeutics 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 
Phone: 626-256-4673 
Fax: 626-301-8981 
e-mail: Clarosa@coh.org 
 
 
STUDY STATISTICIAN: 
 
 
Dongyun Yang, Ph.D. 
Department of Information Sciences 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 
Phone: 626-218-4891 
e-mail: donyang@coh.org 
 
PARTICIPATING SITES: 
City of Hope, Duarte, CA (Lead Institution) 
The Dana-Farber Cancer Institute, MA (Participating Site)          
 MD Anderson Cancer Center, TX (Participating Site) 
COORDINATING CENTER 
Data Coordinating Center 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 
Phone: 626-256-4673 x 63968 
e-mail: DCC@coh.org 
 
AGENT NSC#              NSC#  745100    
 

 
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EXPERIMENTAL DESIGN SCHEMA 
 

 
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PROTOCOL SYNOPSIS 
Protocol Title: 
 
A Phase II randomized, placebo-controlled trial to evaluate the protective function of a CMV-MVA 
Triplex vaccine in recipients of an allogeneic hematopoietic stem cell transplant 
Brief Protocol Title for the Lay Public (if applicable): 
 
CMV-MVA Triplex, a new vaccine to prevent cytomegalovirus infection after allogeneic stem cell 
transplantation 
Sponsor, IND 
COH under IND# 15792 
Study Phase: 
Phase II, Randomized, Blinded, Interventional 
Participating Site: 
City of Hope National Medical Center, MD Anderson Cancer Center and Dana-Farber Cancer Institute 
Rationale for this Study: 
Cytomegalovirus (CMV) is associated with a number of clinical syndromes. [1]. Since the inception of 
allogeneic hematopoietic cell transplant (HCT) as a cure for hematologic malignancies, one of the 
main infectious complications during the first 100 days post-transplant is viremia caused by CMV[2-7]. 
HCT patients are vulnerable to herpes-virus infections, including CMV, as a result of 
immunosuppression associated with treatment strategies aimed at preventing graft rejection or graft 
versus host disease (GVHD) [8-10].  While anti-viral agents that limit viral replication can initially 
control CMV viremia, they are associated with significant toxicity and fail to protect against late-onset 
CMV disease, including reactivation and failure to reconstitute CMV-specific immunity.  Substituting 
toxic antivirals with a vaccine that harnesses the abundant native immune response to CMV may 
improve outcomes for HCT recipients[11]. 
CMV-MVA Triplex is a multiple-antigen recombinant Modified Vaccinia Ankara (MVA) with genes 
encoding 3 immunodominant CMV proteins: UL83 (pp65), UL123 (IE1), and UL122 (IE2). MVA is  
attractive as a therapeutic agent due to its safety record as a smallpox vaccine, including in the young 
and elderly [12]. Recently published data from a randomized, placebo-controlled, double-blind study 
has shown that MVA was safe, well tolerated and immunogenic when used as a vaccine in HCT 
recipients[13]. Additionally, previous results strongly support its use as a vector for delivering 
infectious disease antigens, since MVA vaccination safely induced robust cellular immune responses in 
HCT recipients [13].  
Investigators at COH and collaborating institutions are evaluating CMVPepVax, a novel peptide 
vaccine developed at City of Hope.  A limitation of this peptide-based vaccine is its exclusive use in the 
HLA A*0201 population that only comprises around 30-40% of patients at risk for CMV post HCT. In 
contrast, CMV-MVA Triplex vaccine activity is not limited to an HLA restricted HCT recipient 
population, potentially providing CMV protection to a broader population. Furthermore, MVA 

 
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vaccines such as the CMV-MVA Triplex vaccine have a track record for stimulating potent 
immunogenicity, in most cases far superior than DNA vaccines[17].  
These facts motivated the development of an MVA vaccine candidate incorporating immunogenic 
targets of human CMV.  No current vaccine strategy against CMV developed for HCT recipients uses a 
recombinant vector incorporating multiple cellular response antigens.  The CMV-MVA Triplex vaccine 
is manufactured at City of Hope (COH) Center for Biomedicine and Genetics (CBG), and has been 
tested in a phase I dose escalation study in healthy volunteers. The vaccine was administered twice in 
a 28-day period and shown marked safety in healthy adults vaccinated with up to 5x108 pfu/mL. 
Additionally, CMV-MVA Triplex induced robust expansion of CMV-pp65, IE1 and IE2 CD8 and CD4 T 
cells in the healthy volunteers. There have been no serious adverse events (SAE) or dose limiting 
toxicity (DLT). 
As safety and preliminary immunogenicity have now been established in healthy adults, there is a 
strong rationale to perform a Phase II study to assess safety and efficacy of CMV-MVA Triplex in 
protecting against CMV reactivation and disease in the target HCT patient population at risk of life-
threatening complications. Funding for conducting this Phase II trial is from NCI. 
 
Objectives: 
 
Primary Objectives:  
• Safety: To evaluate the safety and tolerability of CMV-MVA Triplex in vaccinated HCT recipients by 
assessing the following:  non-relapse mortality (NRM) at 100 days post HCT, severe (grade 3-4) acute 
GVHD (aGVHD), and grade 3-4 AEs (CTCAE 4.0) probably or definitely related to the vaccination within 
2 weeks from each vaccination. 
• CMV events: To determine if CMV-MVA Triplex reduces the frequency of CMV events defined as 
reactivation or CMV disease in allogeneic HCT-R+.  A CMV event encompasses - CMV “reactivation” 
(DNAemia ≥500 gc/mL by qPCR, low levels viremia treated by anti-virals; or detection of CMV by 
tissue histology (end-organ disease). 
 
 
Secondary Objectives:  
• Viremia duration and treatment: To characterize CMV reactivation and CMV disease in recipients 
of CMV-MVA Triplex compared to placebo by assessing time to viremia (defined as number of days 
from transplantation to the date of >500 CMV gc/mL), duration of viremia, recurrence of viremia, 
incidence of late CMV viremia/disease (>100 and ≤ 360 days post HCT), use of antiviral drugs 
(triggered by clinically significant viremia of ≥1500 CMV gc/mL), cumulative number of CMV specific 
antiviral treatment days. 
• Transplant-related outcomes: To evaluate the impact of CMV-MVA Triplex on transplant related 
outcomes by assessing the incidence of acute GVHD (aGVHD), chronic GVHD (cGVHD), relapse, non-
relapse mortality, all-cause mortality, infections.  
• Cellular Immunity: To determine 1) if CMV-MVA Triplex increases levels, function and kinetics of 
CMV-specific T cell immunity in vaccinated compared to placebo treated allogeneic HCT-R+, 2) to 
determine whether vaccination induces adaptive NK cell population changes, and increase in the 
highly cytotoxic memory NKG2C+ NK cells, and 3) to explore GVHD biomarkers and compare between 
the vaccine and placebo groups. 
 
 

 
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Study Design: 
Overall design: This is a multisite (COH, Dana-Farber, MDA), randomized, blinded, placebo-controlled, 
parallel-group Phase II trial to evaluate: a) safety and reduction in the frequency of CMV reactivation 
and disease (CMV events) and b) increase in CMV cellular immunity, in allogeneic HCT-R+ vaccinated 
with CMV-MVA Triplex relative to those injected with placebo. This trial is designed to 1:1 randomly 
allocate 102 allogeneic HCT-R+ at risk for CMV complications to CMV-MVA Triplex arm or placebo arm.  
Enrollment and randomization:  
Eligible HCT-R+ will be consented and enrolled pre-HCT. A computer-generated randomization, 
stratified by donor CMV serostatus and center will assign registered participants to the CMV-MVA 
Triplex or placebo arm; the treatment assignment will be generated and provided to pharmacists, 
who are unmasked to treatment-group allocation, in advance of planned vaccination. Enrolled 
participants will be followed for the course of transplant and be assessed for vaccine administration 
criteria for ‘Day-28 post-HCT’ vaccination. Participants who meet vaccine administration criteria will 
receive the vaccine; participants who do not meet criteria will discontinue study participation and be 
replaced.  Only participants who receive a vaccine (CMV-MVA Triplex or placebo) on day 28 will be 
considered “randomized”. Information regarding participants who do not receive vaccine will be 
entered into the computer-generated randomization program to inform the randomization algorithm 
for subsequent treatment assignments. 
Administration of CMV-MVA Triplex /placebo: CMV-MVA Triplex vaccine or placebo is administered 
intramuscularly (IM) on days 28 and 56 post-HCT.  
Duration of participation: Study participation will be completed on Day 365 Post-HCT for all 
participants who receive a vaccine injection. Participants who do not receive a vaccine injection will 
discontinue any further follow-up once the determination is made that the vaccine will not be 
administered.  
Stopping rules:   Formal stopping rules will be implemented, two major safety endpoints; non-relapse 
mortality (NRM) at 100 days post HCT, severe (grade 3-4) acute GVHD (aGVHD), and Serious AEs (SAE, 
grade 3-4) related to the vaccination within 2 weeks from each vaccination will be monitored, but 
based on assessment of individual cases. This study will have an external independent DMC for review 
of protocol events and progress, with reporting of recommendations to COH IRB. 
Endpoints: 
 
Primary endpoints:  
CMV events (reactivation [≥500 CMV genome copies (gc)/mL], use of CMV-directed antivirals, or CMV 
disease) prior to day 100 post-HCT. Timing and recurrence of events are included in the primary 
analysis. 
Key safety endpoints: non-relapse mortality (NRM) at 100 days post HCT, severe (grade 3-4) aGVHD, 
and grade 3-4 AEs (CTCAE 4.0) probably or definitely related to the vaccination within 2 weeks from 
each vaccination. 
Secondary endpoints:  
Viremia treatment and duration:  Duration of viremia, incidence of late CMV viremia (>100 and ≤ 360 
days post HCT), use of antiviral drugs (triggered by clinically significant viremia of ≥1500 CMV gc/mL), 
cumulative number of CMV specific antiviral treatment days. 

 
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Transplant related events: time to engraftment, incidence of aGVHD, chronic GVHD (cGVHD), relapse,   
non-relapse mortality, all-cause mortality, infections.   
Immunological function:   levels and kinetics of CMV-specific T cell immunity, combined with 
immunophenotyping[19-21], and functional studies[22, 23]. NK phenotype and function (cytotoxicity 
and cytokine production). 
Sample Size: 
The target of this Phase II multicenter study is to randomize 102 allogeneic HCT-R+ to either the CMV-
MVA Triplex vaccine arm (N=51), or to the placebo arm (N=51). With the expected drop out rate of 
10-15% prior to randomization, the total accrual is expected to be 113-120.   
Estimated Duration of the Study 
Over the past five years, COH, and Dana-Farber combined have performed >300 adult allogeneic HCT 
procedures annually. We anticipate about 200 to be eligible annually being CMV seropositive. It is 
estimated that accrual will be completed in <2 years from the start date of the trial, and that trial we 
anticipate 1 year of follow up and data analysis.  
Summary of Subject Eligibility Criteria: 
 
Pre-HCT Inclusion Criteria 
• 
Age 18 to 75 years.  
• 
Planned HCT for the treatment of hematologic malignancy (some exceptions including 
multiple myeloma 
• 
Planned related or unrelated HCT, with 8/8 (A,B,C,DRB1) high/intermediate resolution HLA 
donor allele matching and with minimal to no T cell depletion of graft 
• 
CMV seropositive (recipient)  
• 
Seronegative for HIV, HCV and active HBV 
 
Pre-HCT Exclusion Criteria 
• 
Patients undergoing a second allo HCT are not eligible (patients who have undergone a 
previous autologous HCT are eligible) 
• 
Prior investigational CMV vaccine, Experimental anti-CMV chemotherapy in the last 6 months 
• 
No planned use of the following after HCT: Live attenuated vaccines, medically indicated 
subunit or killed vaccines, alemtuzumab or any equivalent in vivo T cell depleting agent, 
medications with known activity against CMV, CMV immunoglobulin  
• 
Aplastic anemia 
• 
Patients with active autoimmune conditions requiring systemic immunosuppressive therapy 
within the previous 5 years are not eligible 
• 
Pregnant women and women who are lactating.  
 
 
Post-HCT Day 28 Vaccine Administration Criteria* 

 
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• 
No ≥ Grade 3 GVHD within 7 days from the day of vaccine 
• 
Disease has not relapsed since HCT 
• 
Successful primary engraftment without secondary graft failure 
• 
No ongoing post-HCT ≥ Grade 3 non-hem AE’s, with the exception of grade 3 glucose 
intolerance, cholesterol, triglyceride, and hyperglycemia 
• 
No for CMV viraemia: CMV qPCR ≤ 500 gc/mL from samples collected and resulted within the 
past 7 days 
• 
Negative for CMV end organ disease (biopsy proven CMV disease) post-HCT 
• 
All prednisone doses within the past 7 days were ≤ 1 mg/kg/day (or prednisone equivalent)  
• 
Not received any prohibited medications (see Section 5.6)  
 
*Participants who do not meet vaccine administration criteria will be replaced.  
Investigational Product Dosage and Administration: 
Participants will be randomized to receive either the CMV-MVA Triplex vaccine or the placebo.  The 
pharmacy will know the randomization status of participants, while the clinical study team and 
participants will remain blinded to the randomization status. 
Approximately 5 x 108 pfu of Triplex Vaccine ( or an equivalent volume of placebo)  will be 
administered IM in the upper arm on Days 28 and 56 post-HCT.  There are no dose modifications, 
although participants must meet vaccine administration criteria in order to receive the vaccine. 
CMV-MVA Triplex vaccine is constituted of 5.1 or 9.1 x108 pfu/mL in PBS containing 7.5% lactose.   
Placebo comprises an isotonic solution of the PBS containing 7.5% lactose.   
Clinical Observations and Tests to be Performed: 
Clinical observations/clinical tests: medical history, physical exams, performance status, routine 
laboratory tests (CBC, chemistry panel), CMV qPCR, GVHD assessment, AE assessment, diagnostics for 
disease relapse assessment (bone marrow and imaging studies), diagnostics for CMV disease 
assessment,  according to institutional standard of care. All of the study endpoints listed above will be 
monitored and recorded. Clinical CMV disease and use of anti-viral drugs will be prospectively 
monitored and recorded. 
Immunologic studies:  Immunologic studies will include monitoring levels, function and kinetics of 
CMV-specific T cell immunity, combined with immunophenotyping studies [19, 21].  The phenotypic 
ratios of CMV-specific T cells will be related to improvement in control of CMV viremia. 
Characterization of highly cytotoxic memory NKG2C+ NK cells[24], linked to CMV reactivation and 
critical for CMV adaptive immune response  will be performed[25-27].   
Statistical Considerations: 
A 40% rate of viremia (CMV reactivation) is expected among unvaccinated, eligible HCT recipients, 
with some possibility of a rate as low as 30%.  Based on the aim to detect either a vaccine-effect 
resulting in a drop from 40% to 15% or a drop from 30% to 10%, we plan to enroll and inject 51 
subjects in each group.  The sample will provide at least 90% power under either scenario, at  a one-
sided 0.10 level of significance (appropriate to this phase II trial).  The sample will allow at least  83% 
power to achieve a 0.05 level of significance. Allowing for 10% of subjects becoming ineligible before 
vaccination, we expect to enroll approximately 114 subjects in 2 years, to vaccinate 102, and observe 

 
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between 20 and 28 reactivation events. 
Analysis Plan:  The primary aim of comparing reactivation event rates will use the time-to-event 
methods incorporating repeated events, as we have previously reported [28].  Computing will be 
done with the coxph function of the R survival package (Therneau) with implements the counting 
process formulation of Andersen and Gill.  Formal stopping rules for safety are embedded in the 
design of this study. Clinical data will be monitored by the PMT and external DMC, and investigation 
of CMV-MVA Triplex will be suspended for safety review if there is evidence of serious treatment-
related AEs.  
 
Specifically: 
(1) 100 days NRM will be monitored as the 12th, 24th and 36th subject on the vaccine arm reaches the 
100 day evaluation point. Operationally, the CRA will notify the monitoring statistician as cohorts of 
24 patients (approximately 12 vaccinated) near the 100 day mark. If NRM frequencies exceed 4, 6, or 
8, at the designated 100 day evaluation point, then the trial will be suspended for safety review by the 
COH, MDA, Dana-Farber  and External DMCs. These numbers were selected to limit the overall false-
alarm probability for this endpoint to less than 0.02 when there is no additional risk due to 
immunization. 
 (2) Severe acute GVHD (aGVHD, grade 3-4) will be monitored as every 12th subject on the vaccine arm 
reaches the 100 day evaluation point. aGVHD will be scored using Keystone consensus criteria[29]. 
The trial will be interrupted at each respective center for safety review by the COH, MDA, Dana-
Farber and External DSMCs if 6 or more of the first 12 recipients, or 9 of 24, or 11 of 36, experience 
Grade 3-4 aGVHD. This would be a significant elevation from the COH/MDA/Dana-Farber historical 
benchmark of 15% of allogeneic HCT recipients with matched sibling donors [30].  These rules are 
determined to limit the overall aGVHD false alarm probability to 0.02 if vaccination does not increase 
risk [30].  
(3) Serious AEs (SAE, grade 3-4) related to the vaccination within 2 weeks from each vaccination will 
be discussed individually by the PMT and will be reported to the DSMC. 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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TABLE OF CONTENTS 
SECTION 
 
 
 
 
 
 
 
 
 
      PAGE 
Experimental Design Schema ......................................................................................................................... 3 
Protocol Synopsis 3 
Table of Contents 10 
List of Tables and Figures .............................................................................................................................. 12 
1.0 
Goals and Objectives (Scientific Aims) ........................................................................................... 14 
2.0 
Background..................................................................................................................................... 15 
2.1 
Introduction/Rationale for Development ........................................................................ 15 
2.2 
Overview and Rationale of Study Design ......................................................................... 20 
2.3 
Preclinical Studies ............................................................................................................ 22 
2.4 
Human Studies ................................................................................................................. 24 
3.0 
Participant Eligibility Criteria .......................................................................................................... 34 
3.1 
Pre-HCT Inclusion Criteria ................................................................................................ 34 
3.2 
Pre-HCT Exclusion Criteria ................................................................................................ 35 
3.3 
Participation of Special Populations ................................................................................ 36 
4.0 
Participant Enrollment and Randomization ................................................................................... 36 
4.1 
Pre-Enrollment Informed Consent and Screening Procedures ........................................ 36 
4.2 
Participant registration .................................................................................................... 36 
4.3 
Randomization ................................................................................................................. 37 
4.4 
Emergency De-Blinding Procedures ................................................................................. 38 
5.0 
Treatment Program ........................................................................................................................ 38 
5.1 
Treatment Overview ........................................................................................................ 38 
5.2 
Maintaining a blinded randomization .............................................................................. 38 
5.3 
Assessments ..................................................................................................................... 39 
5.4 
Criteria for Completing/Discontinuing Study Participation ............................................. 41 
5.5 
Follow-Up and Duration of Participation ......................................................................... 42 
5.6 
Supportive Care, Other Concomitant Therapy, Prohibited Medications ......................... 42 
6.0 
Vaccine Administration Criteria ..................................................................................................... 44 
6.1 
Day 28-Post-HCT Vaccine Administration Criteria ........................................................... 44 
6.2 
Day 56-Post-HCT Vaccine Administration Criteria ........................................................... 44 
7.0 
Data and safety monitoring ............................................................................................................ 45 
7.1 
Definition of Risk Level ..................................................................................................... 45 
7.2 
Monitoring and Personnel Responsible (PMT) ................................................................ 45 
7.3 
AE and UP Definitions ...................................................................................................... 45 
7.4 
Routine Reporting of Adverse Events by Site Investigators ............................................. 47 
7.5 
Expedited Reporting of Unanticipated Problems (UPs) by Site Investigators .................. 47 
7.6 
Independent Data Monitoring Committee (DMC) ........................................................... 48 
7.7 
Toxicities to CMV-MVA Triplex ........................................................................................ 49 
7.8 
Toxicities to the placebo .................................................................................................. 49 
8.0 
Agent Information .......................................................................................................................... 49 
8.1 
CMV-MVA Triplex and placebo –information applicable to vaccine components .......... 49 
8.2 
CMV-MVA Triplex ............................................................................................................. 50 

 
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8.3 
PBS containing 7.5% lactose solution for the placebo ..................................................... 51 
8.4 
Preparation of CMV-MVA Triplex .................................................................................... 51 
8.5 
Preparation of placebo .................................................................................................... 52 
9.0 
Correlative/Special Studies ............................................................................................................ 53 
9.1 
Immunogenicity testing ................................................................................................... 53 
9.2 
MVA vector persistence ................................................................................................... 54 
10.0 
Study Calendar ............................................................................................................................... 55 
11.0 
Endpoint Evaluation Criteria/Measurement of Effect .................................................................... 58 
12.0 
Statistical Considerations ............................................................................................................... 59 
12.1 
Study Design..................................................................................................................... 59 
12.2 
Randomization ................................................................................................................. 59 
12.3 
Sample Size Accrual Rate ................................................................................................. 59 
12.4 
Data Analysis Plan ............................................................................................................ 60 
12.5 
Safety Monitoring ............................................................................................................ 61 
13.0 
Data Handling, Data Management, Record Keeping ...................................................................... 62 
13.1 
Source Documents ........................................................................................................... 62 
13.2 
Data Capture Methods and Management ....................................................................... 62 
13.3 
Case Report Forms/Data Submission Schedule ............................................................... 62 
13.4 
Regulatory Records .......................................................................................................... 63 
14.0 
Adherence to the Protocol ............................................................................................................. 64 
15.0 
Study Oversight, Quality Assurance, and Data & Safety Monitoring ............................................. 65 
15.1 
Site Principal Investigator ................................................................................................ 65 
15.2 
Study Principal Investigator ............................................................................................. 65 
15.3 
Protocol Management Team (PMT) ................................................................................. 65 
15.4 
Monitoring ....................................................................................................................... 66 
15.5 
Quality Assurance ............................................................................................................ 66 
16.0 
Ethical and Regulatory Considerations ........................................................................................... 67 
16.1 
Ethical Standard ............................................................................................................... 67 
16.2 
Regulatory Compliance .................................................................................................... 67 
16.3 
Institutional Review Board ............................................................................................... 67 
16.4 
Informed Consent ............................................................................................................ 68 
16.5 
Women, Minorities, Children, HIV-Positive Individuals (Special Populations) ................ 68 
16.6 
Participant Confidentiality ............................................................................................... 69 
16.7 
Conflict of Interest ........................................................................................................... 69 
16.8 
Financial Obligations, Compensation, and Reimbursement of Participants .................... 70 
16.9 
Publication/Data Sharing ................................................................................................. 70 
17.0 
References ...................................................................................................................................... 72 
Appendix A: Acute GVHD Staging ................................................................................................................. 84 
Appendix B: Chronic GVHD GradinG ............................................................................................................. 85 
Appendix C: Karnofsky Performance Scale ................................................................................................... 86 
Appendix D: Reagents, Equipment and Supplies Necessary for Vaccine Preparation .................................. 87 
Appendix E: SAE/UP Reporting Coversheet .................................................................................................. 88 
Appendix F: Registration Coversheet ........................................................................................................... 89 
 

 
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LIST OF TABLES AND FIGURES 
Figure 1  Construction of CMV-MVA Triplex vaccine…………………………………………………………………………..……………..19 
Figure 2  Potency of CMV-MVA Triplex vaccine in HLA transgenic mice……………………………………………..…………… 23 
Figure 3  In vitro immunogenicity of CMV-MVA Triplex in HCT patients……………………………………………..……………..25 
Figure 4  CMV-MVA Triplex vaccination schedule in healthy adults……………………………………………………..…………….26 
Figure 5  CMV specific responses in healthy adults ……………………………………………………………………………………….28-29 
Figure 6  Levels of pp65 specific T cells post CMV-MVA Triplex vaccination………………………………………..………….….30 
Figure 7  Levels of vaccinia and pp65 specific T cells post CMV-MVA Triplex vaccination……………………………………31 
Figure 8 Vaccinia and pp65 specific T cells in Triplex vacinees with or without prior vaccinina immunity……………32 
Figure 9 Vaccinia virus neutralizing antibodies in CMV-MVA Triplex immunized subjects………….…………….………….33 
 
 
 
Table 1   Adverse events in healthy adults …………………………………………………………………………………………......26 
Table 2   Study Calender ……………..……………………………………………………………………………………………………….….55 
Table 3   Data Submission Schedule ……………..………………………………………………………………………………………….63 
 
ABBREVIATIONS 
Abbreviation 
Meaning 
AE 
Adverse Event 
aGVHD 
BDP 
CBC 
cGVHD 
Acute Graft Versus Host Disease 
Biopharmaceutical Development Program NCI-Frederick, MD 
Complete Blood Count 
Chronic Graft Versus Host Disease 
cGMP 
Current Good Manufacturing Practice  
CLIA 
Clinical Laboratory Improvement Amendments 
CMV  
CMV-MVA Triplex  
Cytomegalovirus 
 Cytomegalovirus-Modified Vaccinia Ankara encoding 3 immunodominant 
CMV proteins 
COH 
City of Hope 
CRA 
Clinical Research Associate/Coordinator 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
CTL 
Cytotoxic T lymphocytes  
DET 
DFCI 
DSMC 
Department of Experimental Therapeutics 
Dana-Farber Cancer Institute 
Data Safety Monitoring Committee 
ECG 
FDA 
Electrocardiogram 
Food and Drug Administration 
FOS 
Foscarnet 
GCP 
Good Clinical Practice 
GCV 
Ganciclovir 
GVHD  
Graft versus host disease  
LCL 
HBV 
Human B-lymphoblastoid cell lines  
Hepatitis B virus 
HCT 
Hematopoietic Stem Cell Transplant 
HCT-R+ 
CMV positive HCT recipients 
HCV 
Hepatitis C virus 
HHV6 
Human herpes virus 6 

 
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HIV 
Human immunodeficiency virus 
HPV 
Human papilloma virus 
HSV 
Herpes simplex virus 
HVTN/DAIDS 
HIV Vaccine Trials Network/Division of AIDS 
ICS 
ID50 
IDS 
IFN 
ICF 
Intra cellular staining 
Median Infective Dose 
Investigational Drug Service 
Interferon 
Informed Consent Form 
IM  
Intramuscular  
IND 
Investigational New Drug  
IRB 
Institutional Review Board 
IV  
Intravenous  
IVS 
In vitro stimulation 
MDA 
MRD  
MUD 
MVA 
NCI 
MD Anderson 
Matched  Related Donor  
Matched  Unrelated Donor  
Modified Vaccinia Ankara 
National Cancer Institute 
NRM 
Non-relapse mortality 
OIDRA 
PBMC 
Office of IND Development and Regulatory Affairs 
Peripheral Blood Mononuclear Cells  
PBS 
PD-1 
Phosphate Buffered Saline 
Program death receptor-1 
pfu 
PI 
R+ 
Plaque forming unit 
Principal Investigator 
CMV positive HCT recipients 
PMT 
Protocol Monitoring Team 
SAE 
Serious Adverse Event 
SAIC 
Science Applications International Corporation  
SOC 
Standard Of Care 
VAL 
Valganciclovir 
 
 
 

 
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1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
Primary Objectives:  
• To evaluate the safety and tolerability of CMV-MVA Triplex in vaccinated HCT recipients by assessing 
the following:  non-relapse mortality (NRM) at 100 days post HCT, severe (grade 3-4) acute GVHD 
(aGVHD), and grade 3-4 AEs (CTCAE 4.0) probably or definitely related to the vaccination within 2 weeks 
from each vaccination. 
• To determine if CMV-MVA Triplex reduces the frequency of CMV events defined as reactivation or 
CMV disease in allogeneic HCT-R+.  A CMV event encompasses any detection of CMV by either qPCR 
(termed “reactivation”: DNAemia ≥500 gc/mL or by tissue histology (end-organ disease). 
 
Secondary Objectives:  
1) To characterize CMV reactivation and CMV disease in recipients of CMV-MVA Triplex compared 
to placebo by assessing time-to viremia (defined as number of days from transplantation to the 
date of >500 CMV gc/mL), duration of viremia, recurrence of viremia, incidence of late CMV 
viremia/disease (>100 and ≤ 360 days post HCT), use of antiviral drugs (triggered by clinically 
significant viremia of ≥1500 CMV gc/mL), cumulative number of CMV specific antiviral treatment 
days. 
2) To evaluate the impact of CMV-MVA Triplex  on transplant related outcomes by assessing the 
incidence of acute GVHD (aGVHD), chronic GVHD (cGVHD), relapse, non-relapse mortality, all-
cause mortality, infections.  
3) To determine 1) if CMV-MVA Triplex increases levels, function and kinetics of CMV-specific T cell 
immunity in vaccinated compared to placebo treated HLA A*0201, CMV seropositive HCT-
recipients, 2) to determine whether vaccination induces adaptive NK cell population changes, 
and increase in the highly cytotoxic memory NKG2C+ NK cells, and 3) to explore GVHD 
biomarkers and compare between the vaccine and placebo groups. 
 
 

 
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2.0 
BACKGROUND 
2.1 Introduction/Rationale for Development 
Human CMV is a double-stranded DNA β-herpes virus which is highly prevalent globally, but rarely elicits 
disease in healthy immunocompetent hosts. The human immune system is unable to clear CMV 
infection and latency, but mounts a robust response targeting multiple immune-evasion genes. CMV is 
among the largest and most complex of known viruses, with a genome encoding around 165 genes [31]. 
The magnitude of the CMV-specific cellular immune response is the most striking aspect of the dynamic, 
life-long interaction between the host and CMV. CMV-specific T cells are essential to control CMV viral 
replication and prevent disease, though do not eliminate the virus or preclude transmission. 
Significant suppression of host antiviral immunity can alter the life-or-death immune surveillance 
homeostasis, allowing CMV reactivation to become detectable or primary infection to cause clinical 
symptoms. Uncontrolled viral replication and dissemination results in the development of life-
threatening end-organ damage (CMV disease)[32-34]. CMV infection is the cause of major complications 
and significant morbidity in the recovery of immune-compromised recipients both at early and late 
times post-HCT [4, 6, 7]. HCT patients are vulnerable to herpes virus infections, including CMV, as a 
result of immunosuppression associated with treatment aimed at preventing rejection or GVHD[35-37].  
Pharmacologic agents used to limit virus replication, such as, GCV or its oral form VAL, and foscarnet 
(FOS) are the methods of choice for prophylaxis CMV infection [4, 38]. Despite this, CMV remains an 
important cause of mortality after HCT diminishing the full curative potential of this successful cancer 
therapy [20, 39]. Furthermore, anti-viral chemotherapy has major side effects, including nephrotoxicity, 
neutropenia, and delayed immune reconstitution which exposes HCT recipients to other opportunistic 
viral, bacterial and fungal infections[7, 40]. For example, the use of GCV/VAL is associated with a higher 
proportion of recipients becoming neutropenic and increased numbers of concomitant fatal infections 
[7, 40]. As GCV/VAL therapy has become ubiquitous in practice, delayed onset of CMV-pneumonia 
(interstitial pneumonitis, IP) is more frequent which suggests that GCV/VAL impairs immunologic 
reconstitution [41, 42]. When antivirals are stopped or when virus resistance occurs, the same disease 
symptoms appear; only frame shifted to ~180 days post-HCT [6, 43-45]. Thus new strategies to control 
CMV are required. Antivirals have limitations, and their use does not address the risks of late-onset CMV 
disease including early CMV reactivation and failure to reconstitute CMV-specific immunity[39].  
Substituting toxic antivirals with a vaccine that harnesses the abundant native immune response to CMV 
may improve outcomes for HCT recipients. In particular, a CMV vaccine that confers protective 
immunity early post-transplant, until normal immunocompetence is re-established in the HCT recipient 
(6 months or earlier post-HCT) may reduce CMV morbidity and the use of antivirals[11].  
MVA: The attenuated poxvirus MVA, engineered with recombinant genes, is being evaluated as a clinical 
vaccine for infectious disease and cancer.  The attractiveness of MVA for clinical use stems from its 
previous safety record as a smallpox vaccine in youngsters and in the elderly[12]. Recent clinical trials 
have affirmed its safety and efficacy in protecting against malaria challenge and generating immune 
responses in humans exposed to the TB bacterium [46, 47].  Lack of viral assembly and avirulence in 
mammals, together with studies showing its safety in heavily immunosuppressed macaques, rodents, 
and in HIV-AIDS patients supports its use in HCT recipients [48-50].  Recently published data from a 
randomized, placebo-controlled, double-blind study [Clinical Trials Registration: NCT00565929] has 
shown that MVA was safe, well tolerated and immunogenic when used as a vaccine in HCT recipients 
[13]. Additionally, they strongly support its use as a vector for delivering infectious disease antigens, 
since MVA vaccination safely induced robust cellular immune responses in HCT recipients [13].  Unlike 
other attenuated poxviruses, the block in viral assembly does not interfere with production of large 
quantities of recombinant proteins in otherwise non-permissive hosts [51].  Multiple sites of foreign 
gene integration in MVA allow the virus to be modifed to expres multiple full-length antigens. These 

 
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facts motivated the development of vaccine candidates incorporating immunogenic targets of human 
CMV.  No current vaccine strategy against CMV that uses a recombinant vector incorporating multiple 
cellular response antigens is being developed for HCT recipients.  Since human CMV infection is severely 
host-restricted, it precludes further evaluation of vaccine candidates to protect against clinical disease in 
animal models. Human testing of vaccines against CMV remains the only valid option to address their 
potential protective function. 
Cellular immune response to CMV in healthy adults: After primary infection, CMV persists under control 
of cell-mediated immune (CMI) surveillance [52-55]. Using whole genome overlapping peptide libraries, 
the targets of the healthy human T cell response to CMV were identified [31, 56]. These studies show 
that the main target for HLA Class 1-restricted T cells is the tegument protein UL83, referred to as pp65 
[57-59]. However, immediate early proteins UL123 (IE1) and UL122 (IE2) also are prominent targets for 
CD8 T cell responses in healthy individuals and patients [31, 60-63]. These 3 targets have a profile that is 
consistent with protective immunity documented in HCT and SOT recipients [20, 64-66]. Phase 1 and 2 
studies showed that pp65- ALVACTM stimulated primary immunity among CMV-negatives to levels 
approaching naturally CMV-positive individuals [67].  Similarly, AlphaVaxTM confirmed that property for 
pp65 and IE1 [68], and TransVaxTM DNA vaccine to a lesser extent [69]. These results provide a rationale 
for our choice of CMV pp65, IE1 and IE2 antigens to be incorporated into the CMV-MVA vaccine. 
CMV immune response in HCT Recipients: CMV interferes with the proper function of the immune 
system [18, 69-75], thus altering the host immune response to CMV. Subunit Ag vaccination strategies 
are the best option to overcome CMV-mediated immunosuppression by limiting viremia, an 
independent predictor of disease [76]. Several reports have associated protection from CMV disease in 
HCT recipients with CMV-specific CD8+ T cell levels between 7-10/µL [18, 71, 77]. IE1-specific T cells 
were found to protect against CMV disease in SOT patients demonstrating the need for both types of T 
cells for efficient protection [65, 66]. COH studies demonstrated that the CD8+ T cell expansions can 
occur prior to d40 post-HCT, and in some cases, these T cells have been traced as clonal expansions 
derived from the donor [72]. Early clinical studies show that CD8+ cytotoxic T lymphocyte (CTL) 
development is necessary to protect from CMV-IP in HCT recipients [3, 11, 78]. In patients who have 
measurable CMV viremia, expansion of CD8+ T cells occurs, especially after viral reactivation [71, 73]. In 
addition, CMV disease occurred only in patients with low level CMV-tetramer+ CD8+ T cells [20, 70, 74]. 
Immunologic monitoring showed the benefit of CMV-positive donors even in the case of CMV-negative 
HCT recipients for long term CMV immune reconstitution [18, 75]. Cellular immunity in HCT recipients is 
augmented during CMV reactivation as shown by increased levels of functional CMV-specific CD8+ T cells 
[20, 70, 71]. In the largest study of its kind, we evaluated impact of T cell memory on the pace of CMV- 
specific immune reconstitution. We confirmed results from multiple clinical trials showing that a CMV-
positive donor enhances CMV immunity in the recipient and decreases severity and duration of CMV 
infection [28, 76, 79-81]. 
Vaccine Strategies against CMV for HCT recipients: The purpose of using live viral vaccination is to 
induce both helper and cytotoxic immunity, which may lead to a durable memory response[82, 83]. 
Plotkin and co-workers established an attenuated strain of CMV, the Towne strain, as a proposed 
therapeutic vaccine in the 1970's, however concerns about using live CMV have minimized its 
applicability[78, 84]. More problematic for using attenuated CMV as a vaccine is the frequency of 
acquisition of a new CMV strain under pre-existing strain-specific natural immunity [85]. Alternative live 
viral approaches for CMV vaccines have focused on canarypox (ALVAC) expressing gB (UL55) which did 
not elicit significant antibody in CMV-negatives [86, 87], or ALVAC-UL83 (PP65) which stimulated robust 
cellular immunity in CMV-negatives equivalent to levels in natural CMV-positives [67]. Further studies 
with ALVAC-UL55 and purified soluble UL55 protein only revealed minimal efficacy, insufficient for 
licensure [88, 89]. Another approach using AlphaVaxTM expressing UL83, UL123 and UL55 [68] showed 
promise when used in healthy adults. However, it is unlikely that AlphaVaxTM can be accepted in HCT, 
since it is based on a recombinant VEE, a live virus that could propagate in humans [68]. TransVaxTM DNA 

 
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vaccine vector expressing either UL55 or UL83 have been evaluated in animal models with good results 
[90-92]. When clinically evaluated, TransVaxTM vaccine, which requires multiple injections showed weak 
immune response in healthy adults, and failed to meet its first endpoint of reduced GCV usage in HCT 
recipients[93, 94]. CMVPepVax, derived from the CMV-UL83 antigen showed remarkable safety and 
elicited vaccine driven immune responses when tested in healthy adults (IRB protocol #03121; J. Zaia, 
P.I.)[15]. CMVPepVax is currently being evaluated in HCT recipients (IRB protocol #12022). In the HCT 
setting, the study indicated safety of injecting CMVPepVax in HCT recipients on day 28 and day 56 post-
HCT, no increase in acute GVHD and reduced CMV reactivation associated with vaccine-stimulated 
immunity [16]. While the CMVPepVax immunologic activity has shown promise, a limitation of the HLA 
A*0201 restricted CMVPepVax is that is solely active in the HLA A*0201 population, that only makes up 
~30-40% of the at-risk HCT population. The CMV-MVA Triplex vaccine being investigated in this protocol 
has a superior track record for stimulating potent immunogenicity than does DNA vaccines, and since it 
expresses whole CMV proteins it has broader recognition and greater applicability for HCT recipients 
than CMVPepVax, which has a narrow applicability. Thus, the CMV vaccine field has made progress since 
the 1970’s, but a clear-cut strategy in which multi- antigen cellular immunity is stimulated from a single 
and safe vaccine vector has been elusive, and it is for these reasons that the development and clinical 
testing of CMV-MVA Triplex vaccine is important. 
 
MVA as a delivery vehicle for vaccines against infectious diseases: The development of MVA as a 
recombinant vaccine stemmed from its benign safety profile as a smallpox vaccine in Europe in the 
1970’s [12, 95, 96].  Development into a vaccine vehicle was only initiated in the early 1990’s, when it 
became clear that non-attenuated poxviruses such as the Western Reserve (WR) strain could not be 
safely administered to immuno-compromised persons [97, 98]. Although MVA is able to efficiently 
replicate DNA in mammalian cells, it is avirulent, because of the loss of two important host range genes 
among >25 mutations and deletions that occurred during its 570 serial passages through CEF [99, 100].  
Despite its restricted host range and inability to produce infectious progeny in human cells, and in 
contrast to NYVAC (attenuated Copenhagen strain) and ALVAC (host range restricted avipox), both early 
and late transcription are unimpaired, making MVA a stronger vaccine candidate [101-104].  In fact, pre-
clinical mouse studies have shown it to be more immunogenic than WR strain, and its ability to be used 
in conditions of pre-existing poxvirus immunity [105, 106]. Consistent with the ability to elicit responses 
against a recombinant MVA transgene product even in the face of pre-existing anti-vaccinia virus 
immunity, repeated administration of recombinant MVA allows re-boosting of responses, despite 
induction of cellular and humoral immune responses against the vector  [107]. This has been reported 
both in Phase I/II therapeutic cancer vaccine trials in which up to 12 recombinant MVA vaccinations 
were given one month apart [108], as well as in a Phase I HIV vaccine trial in which up to three 
recombinant MVA vaccinations were given eight weeks apart [109].   
Studies in rodents and macaques affirm the safety of MVA, including protection against more virulent 
forms of poxviruses in challenge models [110, 111].  After human inoculation, including high risk 
individuals, it is still avirulent without local or systemic reactivity under immunosuppressive conditions 
[104].  Similarly, a therapeutic vaccination with MVA expressing HIV-1 nef demonstrated its safety in 
HIV-1 infected individuals [49].  MVA immunization of malaria-infected adults or those challenged with 
attenuated malaria strains confirmed the safety of the vector and its ability to partially protect against a 
heterologous malaria strain [112, 113].  A trial in France and the USA showed that MVA expressing 
human MUC1 was safe and elicited T cell immunity when given to cancer patients [114].  Recent data 
from a study conducted at the Brigham and Women's Hospital (Boston, MA) by a team of investigators 
from Harvard Medical School and Dana-Farber Cancer Institute have shown that MVA was safe, well 
tolerated and immunogenic when used as a vaccine in HCT recipients[13].  An MVA expressing human 
p53 (p53MVA) developed and characterized at City of Hope [115] has been safely evaluated in a Phase 1 
trial in advanced gastrointestinal cancer patients (IRB #10105)[116].   

 
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In animal disease models, recombinant MVA-based vaccines have either elicited systemic immunity or 
protection against influenza [117, 118], parainfluenza [119], RSV [120], dengue [121], Japanese 
encephalitis [122], malaria [123] and HIV/SIV [124-128].  Recently we used an MVA vaccine similar to 
that proposed in this study. This vector contained rhesus-CMV components and successfully protected 
CMV-negative macaques from infection [129, 130]. This data form the basis for development of CMV-
MVA Triplex vaccine as a vaccine vector for CMV genes.  This study will add to the current body of 
knowledge concerning safety and clinical efficacy of full-length antigens expressed in MVA. 
 
Construction, Expression and Function of 3-antigen MVA Vaccine: Choice of antigens encoded by CMV-
MVA Triplex: 3 CMV gene products, UL83, UL122, and UL123 have been selected as targets for cell 
mediated immune (CMI) responses [61, 131-133].  [See Section 2.1.3 for a definition of these proteins.]  
UL83 has been shown by several groups to be the most immunogenic CMV structural protein (derived 
from the tegument) in terms of human CD8+ and CD4+ T cell responses [57-59, 134-137]. Recently, 
several reports have suggested that the CTL response to UL123 is as vigorous as the response to UL83 
[31, 61-63, 138].  Each of the 3 antigens that are engineered into the multi-protein CMV-MVA Triplex 
vaccine are immunodominant, and their aggregate CMI recognition should exceed 95% of most ethnic 
populations [131].  All 3 antigens elicit memory CTL in humans that both lyse CMV-infected fibroblasts 
in vitro, and are quantifiable using HLA tetramers in fresh PBMC from CMV+ individuals, thereby 
confirming their existence in vivo.  A recent report suggests association of cellular immunity to UL83 
and UL123 with recovery from CMV-retinitis in AIDS patients [139].  The accumulation of T cells specific 
for each of these 3 antigens in individuals with CMV reactivation episodes provides an additional 
justification for their inclusion in a vaccine [140].  Our recent monitoring of the immune response of a 
pre-clinical prototype of the CMV-MVA Triplex vaccine in the PBMC of HCT patients supports its use in 
transplantation [64]. The humoral response to proteins expressed from CMV-MVA Triplex vaccine is 
strong, but there is no evidence that the antibodies neutralize CMV [141].  The majority of the CMV-
neutralizing antibody response has been localized to the gB (UL55) and UL128 gene products [129, 142-
145].  As strong evidence that a humoral component impacts protection against CMV infection after 
HCT is lacking, gB has been omitted from this vaccine.  This CMV-MVA Triplex vaccine strategy focuses 
on the CMI response that has been clearly demonstrated to be associated with protection from disease 
in HCT recipients.   
Functional modification of CMV genes incorporated into MVA: A recent study of the immune response to 
full length UL83 in ALVAC and in TransVax™ DNA vaccine reported no short-term side effects (1-2 years) 
in both healthy volunteers and HCT patients [67, 93, 94]. Investigators have defined regulatory activity 
of the UL123 protein, including trans-activating properties on various cellular promoters [146-148].  
Consequently 85 aa comprising coding exons 2 and 3 have been deleted. Deletion of the two coding 
exons results in a 406-aa protein that is no longer nucleus-associated, but cytoplasmic [61]. The 406 aa 
protein has minimal transactivation activity [147, 148].  Most known CTL epitopes from UL123 are found 
in exon 4, including the identified HLA A*0201-restricted CTL epitopes [62, 63, 138, 149].  Exon5 of 
UL122 was fused in frame to exon4 of UL123 without modification [64]. 
 
Insertion sites, promoter selection and permissive host cells for CMV-MVA Triplex vaccine generation: 
MVA was derived by serial transfer (570 passages) of the parental Ankara strain through chicken embryo 
fibroblasts (CEF) in order to derive a safe alternative to the smallpox vaccine[12].  As a result of its 
adaptation to CEF, several genomic deletions occurred [64, 99, 100].  These adaptations allow MVA to 
freely propagate in CEF to titers exceeding 10e10 pfu/mL, whereas standard mammalian cell lines such 
as CV-1 are non-permissive for propagation.  For the pre-clinical studies conducted under GLP, specific 
pathogen-free (SPF) CEF, obtained from a qualified supplier (Charles River-SPAFAS), were used.  As 
shown in Figure 1, deletion regions referred to as deletion II (del II) and III (del III) were used to insert 
foreign genes by homologous recombination [99].  To obtain the level of foreign protein expression that 

 
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results in a stable virus for manufacturing purposes, the modified H5 (mH5), promoter emphasizing 
early vaccinia gene expression has been utilized in MVA [152], and it provides a powerful boost to 
transcription of foreign gene inserts without causing genomic instability [51, 153] (Figure 2).  The 
recombinant virus was made in two steps; the first being insertion of the pZWIIA plasmid, and the 2nd 
step was insertion of the of the pLW51 plasmid.  The schematic representing construction of the MVA 
vector is shown below in Figure 1. 
 
 
Figure 1: Construction of CMV-MVA Triplex vaccine 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Construction of CMV-MVA Triplex vaccine with transient bacterial marker gene expression: the CMV-
MVA Triplex vaccine was constucted using standard approaches of transfection-infection, purification of 
recombinant viruses and removal of the bacterial marker gene 3 HCMV genes were inserted into MVA 
using established homologous recombination methods [154].  An FDA-certified and traceable seed lot 
from a 1974 isolate was made available by B. Moss (NIAID) that pre-dates the bovine spongiform 
encephalopathy (BSE) outbreak in Europe.  This seed lot is derived from the same batch that was 
approved by the FDA as a smallpox vaccine for use in the by Acambis/Baxter.  Bacterial gene products 
that function as tracking markers in Viral or DNA vectors can cause stimulation of potent immune 
responses [155, 156].  The technology referred to as transient marker stabilization (TMS) which removes 
bacterial gene markers, was initially used to isolate of the CMV-MVA Triplex vaccine [157].  TMS allows 
the first screening to use a color substrate to distinguish the initial insertion as colored plaques.  In 
subsequent rounds, the recombinants are rescreened by PCR and antibody staining to establish excision 
of the bacterial screening marker and presence of the CMV gene product respectively.  Using this 
technology a new plasmid transfer vector (pZWIIA) was developed to enable construction of a marker-
free CMV-MVA Triplex vaccine[158]. The 3-antigen CMV-MVA Triplex vaccine was constructed in three 
stages—the first being the fusion of UL123/e4 with UL122/e5, and verification of its expression and 
immune response [158].  The construction of the 3-antigen vector was furthered by inserting UL123/e4 -
UL122/e5 under the control of the MH5 promoter in pZWIIA.  CMV-pp65 was inserted into a derivative 
of pLW51 in which a single copy of the mH5 promoter drives the expression of the transgene. The final 
vector was extensively characterized for CMV antigen expression, absence of bacterial marker 

 
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sequences, immunogenicity in transgenic mouse strains, and a memory response in human PBMC [64, 
159, 160]. 
The CMV-MVA Triplex has been tested in a phase I dose escalation study in healthy volunteers. Which 
has completed enrollment. The vaccine, administered twice in 28-day period, has shown marked safety 
in healthy adults vaccinated with up to 5x108 pfu/mL. Additionally, CMV-MVA Triplex induces robust 
expansion of CMV-pp65, IE1 and IE2 CD8 and CD4 T cells in vaccinated research subjects. There have 
been no serious adverse events (SAE) or dose limiting toxicity (DLT) and few injection site reactions are 
the only notable AEs.  
Since safety and immunogenicity have been established in healthy adults, there is a strong rationale to 
perform a Phase II study to assess safety and efficacy of CMV-MVA Triplex in protecting against CMV 
reactivation and disease in HCT patients at risk of life-threatening complications.  
 
2.2 Overview and Rationale of Study Design 
This randomized, blinded, placebo controlled, multi-site Phase II safety and efficacy trial will be 
conducted at COH, MDA  and Dana-Farber. This study has been designed to have sufficient statistical 
power for testing clinically significant endpoints. In particular, the efficacy of CMV-MVA Triplex in 
protecting against CMV reactivation and disease in HLA-matched allogeneic (related or unrelated donor) 
HCT R+ (CMV positive HCT recipients) who are at risk for CMV complications[18]. The primary hypothesis 
is that immunizing allogeneic HCT-R+ with CMV-MVA Triplex is safe and will provide superior protective 
benefit compared to a placebo.  The secondary hypothesis is that CMV-MVA Triplex immunizations will 
induce protective levels of CMV cellular immunity in HCT-R+[161-163]. It is anticipated that the trial will 
provide definitive data on vaccine-induced protective immunity against CMV reactivation and disease in 
allogeneic HCT recipients [20].  
Participants must be planned recipients of a first 8/8 HLA-matched[164-167] allogeneic HCT for 
hematological malignancies, CMV-positive, age 18-75 and willing to be monitored for 12 months 
following HCT. Exclusion criteria include receiving T cell depleted HCT, autoimmune disease, HIV, HCV 
and HBV positivity. Excluded diagnoses are aplastic anemia and multiple myeloma. Screening, 
enrollment and informed consent procedures will occur prior to the HCT procedure, as prospective 
participants are considered more able to make an informed decision at this time.  Vaccine 
administration criteria will be assessed on the day of planned vaccination (Day 28 post-HCT). The Day-
28-Post-HCT criteria exclude participants who post HCT have experienced disease relapse, CMV viremia 
or end organ disease, or have received anti-viral treatment or high dose steroids within 7 days of 
planned vaccine administration, or are experiencing ongoing grade 3 toxicities.   
This phase II efficacy trial will enroll and vaccinate 102 allogeneic HCT-R+, randomized to the vaccine 
(N=51) or placebo arm (N=51).  Because approximately 10-15% of enrolled participants may fail to meet 
post-HCT vaccine administration criteria, the total study accrual is expected to be ~115 participants.  A 
computer-generated 1:1 randomization stratified by donor CMV serostatus and center will assign 
participants to the CMV-MVA Triplex or placebo arms. The registrar located at the COH Data 
Coordinating Center will provide treatment assignment to the site pharmacists at COH, MDA or Dana-
Farber, who are unmasked to treatment-group allocation. Because visual inspection of the vaccine at 
the time of administration may reveal the arm to which a participant has been assigned, persons 
administering the vaccine will receive training about the handling of the vaccine in front of the 
participant and colleagues.  All study team members will receive orientation about the importance of 
maintaining a blinded randomization status.  Participants meeting vaccine administration criteria will 
receive injections of either CMV-MVA Triplex or placebo on days 28 and 56 post-HCT, during the critical 

 
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period in which primary CMV reactivation most predictably occurs (~day 40-100)[28, 43]. Both the CMV-
MVA Triplex and placebos are administered in a final 0.9-1mL volume, in the upper arm by 
intramuscular route. 
Previous data, based on safety and immunogenicity in HCT recipients vaccinated with MVA support the 
use of 5x108 pfu as the optimal dose[13].  Additionally, our data from the ongoing Phase Ib trial (COH 
IRB Protocol No. 08173) in healthy adults indicated that CMV-MVA Triplex was safe with limited injection 
site reactions, excellent tolerability and strong immunogenicity when 2 IM injections of 5x108 pfu/mL 
were administered on Day 0 and Day 28.  Thus based on this evidence, a dose of approximately 
5x108 pfu/mL CMV-MVA Triplex administered IM on day 28 and day 56 post-HCT was chosen for use in 
the HCT setting.  Participants in the placebo arm will be injected with an isotonic solution of PBS 
containing 7.5% lactose, the diluent used in the CMV-MVA Triplex preparation.  Participants who do not 
receive the first (Day 28) vaccine will be replaced and removed from the study. All participants who 
receive the first vaccine will be followed and assessed through Day 365 post-HCT, except for participants 
with demonstrated disease relapse.  Participants with relapsed disease will be deemed ineligible upon 
diagnosis, and therefore will be followed for survival only to Day 365.  
HCT recipients are closely observed for safety according to institutional standard of care practices, which 
recommend intense monitoring during the first 100 days following transplant. This study will follow the 
standard of care (SOC) practice till Day 100 post-HCT of weekly or biweekly assessments of CMV qPCR, 
clinical laboratory tests, engraftment assessment, GVHD assessment and physical exam. When clinically 
indicated, CMV disease and disease relapse will be assessed. Disease relapse will also be routinely 
assessed at pre-determined timepoints per SOC. The procedures and clinical care provided to 
participants will not differ from that provided routinely to all post-HCT patients with the exception of 
restricted concomitant medications that could impact study endpoints e.g. prophylactic antiviral 
treatment and T cell depleting agents. 
Between Day 28 and Day 100, study visits will occur bi-weekly to coincide with vaccine administration 
and research blood sample collection, however information relevant to study endpoints can be obtained 
from any clinic visit. After Day 100, an additional four study visits will occur up to and including Day 365 
to ensure periodic documentation and/or evaluation of clinical endpoints and for collection of research 
blood samples.  The study will also investigate the impact of CMV-MVA Triplex on transplant related 
outcomes such as GVHD, disease relapse, mortality, and infection rate.  
Rigorous stopping rules will be implemented, and will include three major safety endpoints: 
• 
non-relapse mortality (NRM) at 100 days post HCT (Note: the rate of non-relapse mortality in 
the allogeneic hematopoetic cell transplant program at City of Hope and DFCI has been ≤10% 
and 12% respectively over the last five years).   
• 
severe (grade 3-4) acute GVHD (aGVHD) 
• 
grade 3-4 AEs (CTCAEv4) related to the vaccination within 2 weeks from each vaccination 
 
CMV-specific immunogenicity (included in the secondary endpoints) will be evaluated in all participants 
in both arms (N=102) every 2 weeks from day 28 until day 100 post-HCT and on days 140, 180, 270, 365. 
Immunologic studies will include monitoring the levels, function and kinetics of CMV-specific T cell 
immunity in vaccinated compared to placebo treated allogeneic HCT-R, combined with 
immunophenotyping studies [19, 21].  The phenotypic ratios of CMV-specific T cells will be related to 
improvement in control of CMV viremia. Based on the encouraging results of the Phase Ib (COH IRB 
08173), levels of pp65, IE1 and IE2-specific T cells associated with protection from CMV viremia and 
disease are anticipated to be detected in a significantly higher proportion of vaccine recipients 
compared to those enrolled in the placebo arm[20]. Additional correlative immunogenicity studies will 

 
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include measuring levels of highly cytotoxic memory NKG2C+ NK cells. These are linked to CMV 
reactivation, are critical for CMV adaptive immune responses and are potentially linked to relapse 
reduction[24]. To investigate the potential impact of CMV-MVA Triplex on GVHD, various GVHD 
biomarkers will be evaluated including: cytokines and hepatocyte growth factor for systemic GVHD [171, 
172],  elafin for skin GVHD [173], regenerating islet-derived 3α (REG3α) for gastrointestinal GVHD[174], 
suppression of tumorigenicity 2 (ST2) for steroid-refractoriness 5 [175] and CXCL9 [176, 177]and B cell–
activating factor (BAFF) for chronic GVHD[176, 177]. The levels of these GVHD biomarkers will be 
compared between the vaccine group and placebo group, as well as between GVHD+ and – groups.   
 
2.3 Preclinical Studies 
2.3.1 
CMV-MVA Triplex vaccine  
Evaluation of the immunogenicity of the CMV-MVA Triplex in mouse models showed that the vaccine 
can stimulate primary immunity against all three CMV antigens (pp65, IE1 and IE2) in both the CD4 and 
CD8 T cell subsets. Since CMV-MVA Triplex activity is not HLA restricted, the vaccine has been 
successfully tested for CMV immune response in various transgenic HLA mouse strains including A2, B7, 
A1 and A11 [64, 160].  Intracellular cytokine secretion (ICS) methods were used to evaluate both CD4 
and CD8 T cells responses to CMV-MVA Triplex vaccine using the peptide library approach applicable to 
any HLA type. We used commercially available pp65 and IE1 peptide libraries (PepMixTM, JPT Peptide 
Technologies GmbH, Berlin, Germany)[72, 140, 178, 179]. An additional peptide library synthesized in 
our laboratory was used to detect responses against IE2 [64]. We were able to detect IFN-γ T cell 
responses using the peptide library approach [28, 64, 140].  Potency of the CMV-MVA Triplex vaccine 
were conclusively demonstrated in HLA transgenic mice [158], as shown in Figure 2.  
 

 
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A. 
B. 
 
 
 
 
Figure 2: Potency of CMV-MVA Triplex vaccine in HLA transgenic mice 
Immunogenicity of CMV-MVA Triplex vaccine in HLA A (A) and HLA B7 transgenic mice (B) as demonstrated by 
specific responses for pp65, IE1 and IE2 post immunization with 50 million pfu of vaccine. The % of IFN-γ producing 
CD8+ T cells (grey bars) and CD4+ T cells (unfilled bars) after stimulation with peptide epitopes or libraries are 
shown. Error bars represent standard error of the mean among the immunized mice (N=3). IFN-γ production in 
mock stimulation was subtracted in all experiments. P values indicate statistically significant differences. 
 
2.3.2 
Animal Toxicology 
The persistence of vectored vaccines is a concern in clinical trials. Whilst the objective is to elicit 
immunity, side-effects due to long-term vector persistence and indefinite immune stimulation must be 
minimized.  The non-recombinant form of MVA has been found to be safe in over 100,00 people [12, 95, 
96] including HCT recipients [13]. However, the recombinant forms are unique and cannot be assumed 
to have an identical safety profile. Therefore pre-clinical toxicology studies of the cGMP-grade CMV-
MVA Triplex Vaccine were conducted in rabbits at the Southern Research Institute (Birmingham, AL; 
Study # 13928.01.01). The vaccinated rabbits showed minimal toxicity, with the CMV-MVA Triplex 
vaccine being cleared from the injection site, blood and all other tissues within 46 days of 
administration.  
 
 
 

 
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2.4 Human Studies 
CMV-MVA Triplex was evaluated in vitro for its ability to stimulate memory responses in PBMC from 
healthy adults and HCT recipients [64, 160]. Interestingly, CMV-MVA Triplex induced strong expansions 
of CMV-specific CD4+ and CD8+ T cell subsets in both healthy adults and patients within 6 months of 
receiving HCT.  Based on the body of pre-clinical and in vitro results the FDA (BB-IND #15792) and IRB 
(08173) granted allowance to proceed with human studies. A Phase Ib clinical trial to evaluate the safety 
and biological efficacy of CMV-MVA Triplex vaccine in healthy volunteers, with or without prior 
immunity to CMV and vaccinia was initiated. This study indicated that CMV-MVA Triplex was safe and 
induced robust expansion of CMV-pp65, IE1, IE2, CD4 and CD8 T cells in 24 healthy volunteers 
(manuscript in preparation).  This safety and immunogenicity data supports initiation of a Phase II trial in 
the HCT setting. 
 
2.4.1 
CMV-MVA Triplex stimulates CMV-specific T cells in human PBMC  
We assessed the in vitro immunogenicity of CMV-MVA Triplex in healthy HCT donor and HCT patient 
blood specimens. CMV-MVA Triplex was first assessed in PBMC from healthy volunteers, providing a 
comparison with HCT patients, which are less well characterized. In both healthy individuals and 
patients there was brisk stimulation of antigen-specific T cell populations following in vitro 
stimulation(IVS) with CMV-MVA Triplex [64]. The memory T cell expansion stimulated by the CMV-MVA 
Triplex for pp65, IE1 and IE2 antigens followed the proportions found ex vivo with the same volunteers 
using the peptide library approach[72, 140, 178, 179]. Moreover they established that CMV-MVA Triplex 
stimulation does not substantially alter the relationship of the T cell subset proportion measured ex vivo 
for all three antigens. We found relationships among the T cell populations to be similar to 
published reports. pp65 promotes a substantial CD4 and CD8 response in over 70% of 
participants, while IE1 and IE2 are recognized less frequently and mainly in the CD8 T cell 
compartment[31, 63]. Interestingly, we found an equivalently strong recognition of CMV-MVA Triplex in 
HCT recipients, which in some cases was more vigorous than in the PBMC of healthy adults. Figure 3 
shows the magnitude of the CMV-specific T cell responses in HCT patients. The T cell response was more 
pronounced in the CD8 population compared to the CD4 T cells, which is reflective of the ex vivo profile. 
 
 

 
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Figure 3: In vitro immunogenicity of CMV-MVA Triplex in HCT patients 
CMV MVA Triplex stimulates CMV-specific T cells in PBMC from HCT recipients after IVS. A comparison was made 
between the ex vivo level (grey boxes) versus post-IVS for each stimulation. PBMC were divided into four aliquots 
and were individually co-incubated with pp65 (black), IE1 (white), IE2 (striped) peptide libraries or peptide diluent in 
single use aliquots. Standard gating procedures were employed for each individual flow acquisition, such that 
conditions were standardized for all evaluations. The plots show the percentage of IFN-γ producing T cells for each 
antigen-specific peptide library.  
 
2.4.2 
Phase Ib clinical trial in healthy adults (IRB 08173) 
CMV-MVA Triplex vaccine was further evaluated in a Phase Ib clinical trial (IRB Clinical Protocol No. 
08173), conducted in healthy adults to assess safety and immune response. To establish safety of CMV-
MVA Triplex vaccine, a dose escalation study was performed in healthy volunteers (COH employees), 
≥18 and ≤60 years of age, either CMV seropositive or seronegative.  The study treated 8 subjects at 3 
dose levels: 1x107 pfu/dose, 5x107 pfu/dose, and 5x108 pfu/dose.  Volunteers received two IM injections 
of 1mL volume in the upper non-dominant arm, each at the same dose over a 4-week period (see Fig 4).  
 
 

 
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2.4.2.1 Safety of CMV-MVA Triplex vaccine – primary endpoint 
Among the twenty-four healthy volunteers (HV) vaccinated with CMV-MVA Triplex Vaccine at DL1, DL2 
and DL3 few local site injections were reported.  Enrollment has been completed, with none of the 
patients experiencing SAEs or DLT or withdrawing from the study. Thus, the first in human study of 
CMV-MVA Triplex vaccine has met the primary objective, showing an excellent safety profile (Table 1). 
 
 
                      Table 1: AEs  in healthy adults trial (Phase Ib trial) 
Adverse Events: Since rare occurrences of carditis with heart failure has occurred in human studies using 
a replicating vaccinia virus, the FDA recommended that all volunteers on this study be monitored for any 
change in electrocardiogram and cardiac troponin after vaccination[180].  No change was reported for 
all CMV-MVA Triplex vaccinated volunteers and no other cardiac AE were reported. Our results are in 
agreement with a recent systematic review, which reported cardiac safety surveillance from 6 Phase I 
trials of MVA vaccines. No detection of cardiac adverse reactions in any study participants immunized 
Figure 4:  CMV-MVA Triplex 
vaccination schedule in healthy 
adults trial (Phase Ib) 

 
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with MVA was found. Unlike a replicating vaccinia virus, MVA is a highly attenuated and does not 
replicate in the human body[181]. A recent trial has shown that MVA was safe with no cardiac AE when 
used as a vaccine in HCT recipients, who were examined closely for vaccine induced cardiac side 
effects  [13]. Additionally, an MVA expressing human p53 (p53MVA) developed by the Department of 
Experimental Therapeutics (DET)[115] was administered to 12 advanced gastrointestinal cancer patients 
in a Phase 1b study, with no SAEs observed (IRB #10105)[116].  Persistence of Viral DNA: Persistence of 
the CMV-MVA Triplex vaccine in healthy volunteers was monitored in accordance with FDA 
recommendations.  If MVA DNA is detectable, monitoring in the study subject concerned is continued, 
up to a maximum of 12 months. The method used is based on an assay developed for assessing the 
presence of MVA sequences during the derivation of the vaccine at COH and was validated by the 
Quality Assurance Department of COH. This real-time PCR approach employs separate sets of primers 
for the MVA backbone and the CMV insert genes utilizing TaqMan™ reagents. The assay is sensitive to  
<20 copies of MVA DNA per sample and can be used to detect low level residual MVA in blood and other 
tissues. A plasmid DNA standard was employed to quantify the copy number of MVA and insert genes 
detected blood specimens.  Blood and tissues from the CMV-MVA Triplex vaccinated rabbits (described 
in section 2.3.2) were tested in this way, and the results reported to the FDA as part of the BB-IND-
13792 submission. From this successful submission, permission to proceed to the Phase I trial was 
granted.  Persistence of the CMV-MVA Triplex vaccine was monitored in healthy volunteers after 
administration of both doses of vaccine, according to the methodology described above.  Measurements 
were conducted in triplicate with one additional sample spiked with 50 copies of vaccine DNA. DNA from 
the dose level 1 and 2 cohorts showed undetectable levels of MVA DNA, or values below the detection 
limit of the assay. In the Dose Level 3 cohort, low levels of MVA DNA above the assay detection limit 
were  detected at day 90, in 2  out of 8 of the vaccinated subjects. Since no MVA DNA was detected in 
these  subjects blood at previous time points, these  could be  sporadic, anomalous results. Considering 
the high doses of CMV-MVA vaccine administered to these subjects, the low level of MVA DNA detected 
in these   subjects, at a single time point, is not a cause for concern. Nonetheless, the day 360  time 
point will be monitored to confirm complete disappearance of MVA DNA. 
2.4.2.2 CMV-MVA Triplex Induced Immune Responses – secondary endpoint 
CMV-MVA Triplex vaccine driven immune responses are robust in DL1 (N=8) and DL2 (N=8) healthy 
volunteers (Figure 5). Most immunized volunteers showed post-vaccination increases in CMV pp65, IE1- 
and IE2- specific T cell levels. The most significant values were observed in CMV positive subjects who 
had low levels of CMV-specific T cells pre-vaccination (Figure 5). We evaluated the cellular CMV-specific 
response by measuring CD137 on the surface of CD8 and CD4 T cells after 24 hours stimulation with 
pp65, IE-1 and IE2 peptides[182]. CD137 is expressed only on recently activated T cells, and its 
expression correlates with functional activation of T cells [183, 184]. We have previously shown that the 
frequency of CMV-specific T cells producing IFN-γ were lower than those expressing the CD137 marker 
[182, 184]. This suggests that T cell responses against CMV might not be fully characterized with a single 
functional cytokine assay. However, an antigen specific activation marker such as CD137, which is 
associated with multiple T cell functions, may provide a superior signal for CMV immune-monitoring in 
the transplant setting[185, 186]. Statistical comparisons (rank-sum test) indicate that average post-
vaccination levels of pp65, IE1 or IE2 specific CD8 and CD4 T cells were significantly higher than baseline, 
with p-values ranging from 3 x 10-5 to 0.025.  For example, pp65 stimulated CD4+ CD137+ T cells rose 
from a pre-vaccination median of 1.3 cells/µL to a post-vaccination median of 4.4 (p = 3 x 10-5) and pp65 
stimulated CD8+ CD137+ T cells rose from a pre-vaccination median of 0.22 cells/µL to a post-
vaccination median of 3.1 (p = 0.003). 

 
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Figure 5: Pre- and post-vaccination ex vivo CMV specific responses in HV immunized with 
CMV-MVA Triplex: Levels of CD137+ CMV specific T cells in DL1 and DL2 cohorts. Each line shows the 
kinetics of the vaccine response with pre- and post vaccination levels of CD8 or CD4 T cells specific for pp65 IE1 
and IE2 peptide libraries.  
 
pp65 
                DL1 Summary                                   DL2 Summary                                     DL3 Summary 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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IE1 
           DL1 Summary 
                            DL2 Summary                                   DL3 Summary 
 
 
 
     
 
 
 
 
 
IE2 
 
 
 
 
 
 
 
 

 
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In addition, T cells specific for the immuno-dominant  pp65495-503 cytotoxic T cell epitope were monitored 
using Dextramer technology (Immudex, Copenhagen, Denmark), and were found to significantly 
increase post-vaccination. Interestingly, the response patterns to this epitope were similar to those 
detected against the whole pp65 peptide library (see Figure 6 below). These results are consistent with 
multiple findings showing the immune dominance of pp65 HLA A*0201 epitope and indicate that CMV-
MVA Triplex does not alter the T cell recognition patterns of immunodominant CMV antigens measured 
ex vivo [15, 31, 52]. 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 6: Ex vivo levels of pp65 specific T cell in an HV immunized with CMV-MVA Triplex:  
Pre- and post-vaccination levels of CD8 T cells specific for either the whole pp65 protein (left y axes, using 
pp65 library, red symbols) or the HLA A*0201 pp65495-503 CTL epitope (right y axes, using pp65495-503 
Dextramers®, black symbols), in representative UPN 5 DL1.   
 
In conclusion, these preliminary results indicate CMV-MVA Triplex vaccine is strongly immunogenic in 
healthy adults even with the lowest dose of vaccine administered (107 pfu/mL), and that it does not alter 
the T cell recognition patterns from what has been found in naturally CMV infected healthy adults 
 
Poxvirus Specific Immune Responses 
All vaccinated volunteers were evaluated for poxvirus immunity after CMV-MVA Triplex vaccination. 
These evaluations were performed to assess the recognition levels of the MVA viral vector in healthy 
volunteers immunized with CMV-MVA Triplex vaccine. 
 
 
 

 
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Cellular immune responses 
Vaccinia virus specific T cells levels were measured by CD137 assay [182] using peptide arrays composed 
of HLA supertype A and B epitopes of vaccinia virus proteins (obtained from NIH Biodefense and 
Emerging Infections Research Resources Repository, Manassas, VA). Post vaccination increases in 
vaccinia specific T cells were frequently detected in the peripheral blood of CMV-MVA Triplex vaccinated 
volunteers. Increased vaccinia virus-specific T cells were generally higher after the 2nd immunization and 
more conspicuous for CD8 than the CD4 T cell subset, as the vaccinia peptide arrays were mainly 
composed of CD8 T cell epitopes. Levels of vaccinia specific T cells vary among subjects. Interestingly, 
they were often of comparable magnitude to T cell levels elicited against the pp65495-503 CTL epitope, but 
lower than the response to the whole CMV pp65 peptide library. Moreover they followed the kinetic 
patterns similar to those of the CMV immune responses (Figure 7). 
 
 
 
 
 
 
Figure 7 Ex vivo levels of vaccinia and pp65 specific T cell in a healthy volunteer immunized 
with CMV-MVA Triplex:  Pre- and post-vaccination levels of CD8 T cells specific for the vaccinia peptide library 
(red line) compared with levels specific for the pp65495-503 CTL epitope (left panel, black dotted line) and the whole 
pp65 peptide library (right panel, black dotted line) in representative UPN 2 DL1.   
 
 
In the DL1 vaccine cohort four volunteers were born before 1973, while the remaining subjects were 
born on or after 1973. 1973 was the final year of smallpox immunization in the U.S. [187] and these four 
subjects had received smallpox vaccination. However, their previous immunity with vaccinia did not 
result in marked differences in the T cell responses compared to vaccinia naïve subjects (see Figure 8). 
Our results are in agreement with previous longitudinal studies  in which antiviral antibody responses 
were found to be highly stable and could be present 1 to 75 years post-vaccination, while anti-vaccinia T 
cell responses decreased 8 to 15 years after the last vaccination[187, 188]. 

 
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Figure 8: Ex vivo levels of vaccinia specific T cells in CMV-MVA Triplex vacinees with or without prior 
vaccinia immunity Pre- and post-vaccination levels of CD8 T cells specific for the vaccinia peptide library in 8 
heathly volunteers, including UPN 5 who was vaccinia naïve at the time of Triplex vaccination (year of birth 1990) 
and UPN 8 who had previously received smallpox vaccination (year of birth 1968).   
 
 
Humoral response   
Vaccinia neutralization assays were performed with a modification of a published protocol[189] using an 
MVA expressing the fluorescent marker Venus [129]. Briefly, serial 5-fold dilutions of sera, starting with 
a 1:20 dilution, were incubated with 0.5 × 106 pfu of venus fluorescent-expressing vaccinia virus.  
Subsequently human B-lymphoblastoid cell lines (LCL) were added at a concentration of 0.5 pfu per cell 
in 96-well plates. Fluorescence intensity in infected cells was compared with wells containing cells and 
virus, but no serum (positive control) and cells and serum containing no virus (background control). In 
addition, anti-rabbit vaccinia purified serum anti-vaccinia rabbit sera (a kind gift from Dr. Mary 
Marovich, Henry Jackson Foundation, Bethesda, MD) was used as an internal reference standard.  
Neutralization antibodies specific for vaccinia virus were raised in the large majority of the volunteers 
immunized with CMV-MVA Triplex vaccine. Levels rose after the second injection and remained stable 
until the study was concluded. As expected, all 4 study volunteers from DL2 who were born before year 
1973 did have detectable vaccine virus neutralizing antibody before vaccination with CMV-MVA Triplex 
vaccine (Figure 9). 
 

 
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Figure 9: Levels of vaccinia virus neutralizing antibodies in CMV-MVA Triplex vaccine 
immunized subjectsfrom the DL2 cohort 
The ID50 titer was calculated as the serum dilution that caused a 50% reduction in venus expression (green 
fluorescence) on the vaccinia-infected LCL compared to the virus+LCL control samples after subtraction of 
uninfected LCL control. A positive response for the CMV-MVA Triplex vaccine was defined as a titer ≥ 2 times the 
baseline (day 0) titer and ≥ 1:20. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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3.0 
PARTICIPANT ELIGIBILITY CRITERIA 
3.1 
Pre-HCT Inclusion Criteria 
Participants must meet all of the following criteria on screening examination to be eligible to participate 
in the study:  
Informed Consent and Willingness to Participate 
___ 1. All subjects must have the ability to understand and the willingness to sign a written 
informed consent. 
___ 2. Participant must be willing to comply with study and/or follow-up procedures, including 
willingness to be followed for one year post-HCT. 
Age Criteria 
___ 3. Age 18 to 75 years.  
Nature of Illness and Transplant Related Criteria 
___ 4. Planned HCT for the treatment of the following hematologic malignancies: 
o Lymphoma (Hodgkin and Non-Hodgkin) 
o Myelodysplastic syndrome 
o Acute lymphoblastic leukemia in first or second remission (For Acute Lymphoblastic 
Leukemia/Lymphoblastic Lymphoma, the disease status must be in hematologic 
remission by bone marrow and peripheral blood. Persistent lymphadenopathy on CT 
or CT/PET scan without progression is allowed.) 
o Acute myeloid leukemia in first or second remission 
o Chronic myelogenous leukemia in first chronic or accelerated phase, or in second 
chronic phase 
o Other hematologic malignancies including chronic lymphocytic leukemia, 
myeloproliferative disorders and myelofibrosis. Patients with multiple myeloma and 
those with non-malignant disease such as aplastic anemia are excluded* 
 
*Adult cases of multiple myeloma (MM) are excluded as allogeneic HCT is not standard of care for MM, 
and is only performed in very advanced cases with an associated high risk of relapse and NRM. Adults 
with aplastic anemia are excluded because their standard management includes T cell depletion with 
agents such as ATG, which is not permissible on this protocol (see Section 3.2 point 6).  Patients 
undergoing a second allo HCT are not eligible (patients who have undergone a previous autologous HCT 
are eligible). 
 
___ 5. CMV seropositive (recipient) 
___ 6. Planned related or unrelated HCT, with 8/8 (A,B,C,DRB1) high/intermediate resolution 
HLA donor allele matching 
___ 7. Planned HCT with minimal to no-T cell depletion of graft 
___ 8. Conditioning and immunosuppressive regimens according to institutional guidelines are 
permitted 

 
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Clinical laboratory parameters  
___ 9. Negative serum or urine β-HCG test (female patient of childbearing potential only) 
within two weeks of registration. 
___ 10. Seronegative for HIV, HCV and active HBV (Surface Antigen Negative) within 2 months of 
registration. 
 
Child Bearing Potential 
___ 11. Agreement by females of childbearing potential and sexually active males to use an 
effective method of contraception (hormonal or barrier method of birth control or 
abstinence) prior to study entry and for up to 90 days post-HCT. Should a woman 
become pregnant or suspect that she is pregnant while participating on the trial, she 
should inform her treating physician immediately.  
 
3.2 Pre-HCT Exclusion Criteria 
Prospective participants who meet any of the following criteria will not be eligible for admission into the 
study: 
Previous therapies 
___ 1. Any prior investigational CMV vaccine 
___ 2. Experimental anti-CMV chemotherapy in the last 6 months 
 
Planned medications from the time of HCT to day 70 post-HCT 
___ 3. Live attenuated vaccines  
___ 4. Medically indicated subunit (Engerix-B for HBV; Gardasil for HPV) or killed vaccines (e.g. 
influenza, pneumococcal, or allergy treatment with antigen injections) 
___ 5. Allergy treatment with antigens injections  
___ 6. Alemtuzumab or any equivalent in vivo T-cell depleting agent 
___ 7. Antiviral medications with known therapeutic effects on CMV such as GCV/VAL, FOS, 
Cidofovir, CMX-001, maribavir. Acyclovir has no known therapeutic efficacy against CMV 
and is allowable as standard of care to prevent HSV.  
___ 8. Prophylactic therapy with CMV immunoglobulin or prophylactic antiviral CMV treatment  
___ 9. Other investigational product – concurrent enrollment in other clinical trials using any 
IND drugs with unknown effects on CMV or with unknown toxicity profiles is prohibited. 
___ 10. Other medications that might interfere with the evaluation of the investigational 
product  (see Prohibited Medications, Section 5.6) 
Other illnesses or conditions 
___ 11. Patients with active autoimmune conditions requiring systemic immunosuppressive 
therapy within the previous  5 years are not eligible 
___ 12. Pregnant women and women who are lactating. CMV-MVA Triplex risks to pregnant 
women are unknown.  Because there is an unknown but potential risk for adverse 

 
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events in nursing infants secondary to treatment of the mother with the administered 
vaccine, also breastfeeding should be discontinued if the mother is enrolled on this 
study.   
___ 13. Any other condition that would, in the Investigator’s judgment, contraindicate the 
patient’s participation in the clinical study due to safety concerns or compliance with 
clinical study procedures, e.g., social/ psychological issues, etc. 
 
 
Noncompliance 
___ 14. Prospective participants who, in the opinion of the investigator, may not be able to 
comply with all study procedures (including compliance issues related to 
feasibility/logistics). 
 
3.3 Participation of Special Populations 
A discussion of the inclusion, exclusion, and representation participation of women, minorities, children 
and HIV positive individuals is provided in Section 16.5.  
 
4.0 
PARTICIPANT ENROLLMENT AND RANDOMIZATION 
4.1 Pre-Enrollment Informed Consent and Screening Procedures 
Diagnostic or laboratory studies performed exclusively to determine eligibility will be done only after 
obtaining written informed consent.  Studies or procedures that are performed for clinical indications 
(not exclusively to determine study eligibility) may be used for baseline values and/or to determine pre-
eligibility, even if the studies were done before informed consent was obtained. The informed consent 
process is to be fully documented, and the prospective participant must receive a copy of the signed 
informed consent document.  See Table 10 footnote f and Section 16.4 for more information regarding 
informed consent. Screening procedures are listed in Section 10, Table 10 Study Activity Calendar.  
4.2 Participant registration 
4.2.1 
COH DCC Availability and Contact Information 
Eligible subjects will be registered on the study centrally by the Data Coordinating Center (DCC) at City of 
Hope.  DCC staff are available between the hours of 8:00 a.m. and 5:00 p.m. PST, Monday through 
Friday (except holidays).   DCC contact information is as follows: 
• 
phone: (626) 256-4673 ext. 63968 
• 
e-mail: DCC@coh.org 
4.2.2 
Slot verification and reservation 
As the study nears completion of accrual, study team personnel (including physicians, protocol nurses 
and/or CRCs) may wish to contact the DCC to verify slot availability and to reserve an open slot or be 
placed in queue for slot opening.  Slots may only be held for a limited time which will be determined by 
the PMT. The Data Coordinating Center should be notified of cancellations of prospective participants 
holding slots as soon as possible. 

 
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4.2.3 
Registration procedure 
To register a participant, the subsequent procedure is to be followed. 
1. The participating site’s data manager/coordinator/research nurse should contact the DCC 
via telephone or email to provide notification regarding the pending registration and 
communicate desired timeline of the registration, especially if it must be completed 
promptly 
to 
meet 
the 
registration 
window 
(60 
days 
to 
0                             
days before planned HCT). 
2. The data manager/coordinator/research nurse should then e-mail copies to DCC@coh.org  
of the following documents to the DCC:  
o Registration Cover Sheet (Appendix F or G) 
o Completed Eligibility Criteria List  
o Source documentation to support eligibility criteria**  
o Signed informed consent document (if permitted by institutional policy) 
o Signed HIPAA authorization form (if separate from the informed consent document) 
o Signed subject’s Bill of Rights (COH only)  
**For COH participants, provide copies of source documentation only if not readily 
available as a finalized record in the COH EMR  
3. After having received all transferred documentation, the DCC will review the documents to 
verify eligibility, working with the participating site as needed to resolve any missing 
required source elements. A subject failing to meet all protocol eligibility requirements will 
not be registered.  
4. Once eligibility has been confirmed, DCC staff will register the participant by: assigning a 
subject accession number, register the subject on study centrally into MIDAS for  MD 
Anderson and Dana-Farber participants (the COH CRC will directly accession into MIDAS), 
and enter the subject into the eCRF system, Medidata RAVE. 
5. Once registration has been completed, DCC staff will send a Confirmation of Registration 
Form, including the participant study number and planned date of HCT procedure to:  
o the site study team: site PI, treating physician, protocol nurse, CRC and pharmacy 
o the sponsor team: (Drs. Nakamura, Aldoss, La Rosa, and the COH CRC) 
 
4.3 Randomization 
The treatment assignment will be masked from patients and health care providers, but known to the 
DCC coordinators, study monitors, auditors, pharmacy personnel, and the statistician.   See Section 5.2 
for additional information about maintaining a blinded randomization. 
The DCC staff will use a computer-generated randomization , stratified by donor CMV serostatus and 
center to assign registered participants to the CMV-MVA Triplex or placebo arm. The treatment 
assignment will be generated and provided to pharmacists in advance (usually ~day 21 post-HCT) of 
planned vaccination. The DCC staff will confirm that the treatment assignment was received by the 
pharmacy.  
The DCC specialist will request a copy of source documents regarding donor CMV serostatus, if not 
already provided/available. The DCC staff may contact the study team coordinator to confirm the HCT 
procedure did occur prior to generating the treatment assignment. 

 
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As soon as the study team is aware that a registered participant will NOT meet the criteria to receive the 
initial vaccine, this should be promptly communicated to the DCC.  This information will be entered into 
the computer-generated randomization program to inform subsequent treatment assignments. 
Only participants who receive a vaccination (CMV-MVA Triplex or placebo) on day 28 will be considered 
“randomized”.   
 
4.4 Emergency De-Blinding Procedures 
Participant’s randomization status will be un-blinded in the event a patient on this study develops a life-
threatening toxicity or serious adverse event for which the participant’s physician or other health care 
professional feels that it is in the patient’s best interest to know the randomization status of the 
participant. 
The following procedure should be followed:  
Information regarding the rationale for de-blinding will be provided to the Site PI, the COH DCC, 
and the Study PI.  
The Site PI will communicate with Pharmacy and will provide authorization to un-blind 
patient’s treatment.  
In this very unlikely event, the PMT will determine if and how the de-blinding should impact the 
participant’s continued participation in the study or analysis of collection points post de-blinding. This 
plan will be provided to the IRB of record and the COH IRB and DSMC as per COH institutional 
requirements. The date and reason for de-blinding must be noted in the medical record and captured in 
the eCRF. 
 
 
5.0 
TREATMENT PROGRAM 
5.1 Treatment Overview 
The study intervention will consist of a vaccine administration of either CMV-MVA Triplex (N=51) or 
placebo (N=51), depending on participant randomization.  
Participants will receive vaccine administration on days 28 and 56 post-HCT if vaccine administration 
criteria are met (Sections 6.1 and 6.2) and confirmed by the DCC (who will notify the study team 
pharmacy of the confirmation). 
Participants who do not receive the Day 28 Post HCT vaccine administration will be replaced. 
Participants who receive at least one vaccine administration will complete all procedures detailed in 
Section 10, except for participants who experience relapsed disease for whom all post-relapse 
assessments will cease and will be followed only for survival through day 365-Post-HCT.  
Windows for vaccine administration are detailed in Section 10, Study Activity Calendar. 
 
5.2 Maintaining a blinded randomization 
The treatment assignment will be masked from patients and health care providers, but known to the 
DCC coordinators, study monitors, auditors, pharmacy personnel, and the statistician. Due to differences 
in appearance of the active agent and the placebo, the person administering the vaccine (placebo or 
active agent) should limit discussions regarding the characteristics regarding the agent’s appearance or 

 
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administration (i.e. ease or difficulty of administration) to the study pharmacist. Since the person 
administering the vaccine may detect a difference in appearance between placebo or active agent, 
he/she will be furthermore excluded from performing any protocol-required procedures or providing 
day-to-day medical care of the participant for the duration of the trial. The pharmacist will keep the 
treatment assignments and accountability documentation such that the documents cannot be accessed 
by the individuals who conduct protocol-required assessments, follow-up assessments, or those 
involved in the day-to-day medical care of the subjects during the trial. Nicola Hardwick will be the 
designated un-blinded PMT contact for Pharmacy. Disclosure of any knowledge of the randomization 
status to persons other than those permitted to know the randomization status would result in a 
protocol violation.  
 
5.3 Assessments 
Patients undergoing HCT are heavily monitored for safety according to institutional SOC practices. The 
following assessments will occur for safety and/or endpoint analysis with the schedule indicated in 
Study Activity Calendar (Section 10), per institutional SOC, and as clinically indicated.  
5.3.1 
Post-Vaccination Assessment 
Vaccine and placebo injections will be administered intramuscularly. All subjects will be monitored for at 
least 30 minutes after each immunization for any local or systemic reactions including vital signs 
(temperature, pulse, blood pressure, and respiratory rate). Notation will be made of the subject’s 
temperature and of any local reaction at the injection site.  
 
5.3.2 
GVHD assessment and performance status 
Acute GVHD will be assessed and graded according to the Keystone Consensus grading system 
(Appendix A). Chronic GVHD will be classified per Appendix B by type of onset (progressive, interrupted, 
de novo, or chronic); basis of diagnosis (histologic/biopsy proven, clinical evidence, both, or unknown); 
Limited or Extensive chronic GVHD; and overall severity of GVHD (mild, moderate, or severe). 
Performance status will be evaluated utilizing the Karnofsky Performance Scale (Appendix D).  
5.3.3 
CMV monitoring 
For CMV monitoring, standard qPCR clinical laboratory methods will be used to evaluate CMV viral load 
and possible vaccine failure at least weekly (usually twice weekly), or as required by SOC until day 100. 
The methods used at the two study sites are as follows:  
COH:  Focus 3M Integrated Cycler and Simplexa CMV Kit for in vitro diagnostic use, detection limit 250 
gc/ml, reported in WHO IU/ml (conversion factor 2.5; 250gc/ml=625IU/ml, note all PCR values in this 
protocol are expressed by gc/ml based on the COH assay). 
DFCI: Roche Cobas CMV VL assay, detection limit 150 gc/ml, one copy equivalent to 0.91 WHO 
International Units (IU).  
MDA:  Cobas CMV PCR, detection limit 97 CMV IU/mL, with a reportable range >137 IU/mL.  
Based on available information these methods are considered sufficiently similar. However, selected 
samples from DFCI study subjects will be stored for batch testing at COH to confirm equivalence of the 
two methods (see study calendar for details). 
Clinical CMV disease status will be documented at each study visit, which may include the absence or 
presence of suspected CMV disease.  When clinically indicated and per SOC, CMV disease will be 
assessed and, when present, the site (upper GI, lower GI, other, specify) and method of detection in the 
tissue (tissue culture, pathology etc.) will be documented. Presentations or suspected presentations of 

 
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CMV disease in the absence of qPCR >500 gc/mL will be evaluated by the treating investigator in 
conjunction with the blinded PMT before a determination is made.  
5.3.4 
Engraftment assessment 
Engraftment will be assessed by monitoring the recipient’s absolute neutrophil count. The date of 
engraftment is defined as the first date of 3 consecutive  laboratory values obtained on different days 
when the peripheral blood absolute neutrophil count is ≥ 500/mm3; for the purposes of recording into 
the case report form, the date of engraftment can be derived from the ANC values in the clinical 
laboratory results so long as there is accompanying documentation in the medical record that 
engraftment did occur.  
Graft failure following engraftment (secondary graft failure) is defined as a fall in the absolute neutrophil 
count below 500/mm3 for 3 or more consecutive laboratory values  following initial engraftment that is 
not due to disease relapse/progression, infection or secondary medication effect; the date of graft 
failure will be defined as the date when the criteria for graft failure are confirmed by the clinician-
investigator. 
5.3.5 
Disease relapse 
Disease relapse will be assessed (including timing of assessment)  according to institutional SOC practice 
for the participant’s specific hematologic malignancy. At defined clinic visits, the disease relapse status 
should be documented, which may include the presence or absence of clinical signs of disease relapse. 
Relapse is defined as  “morphologic relapse” for acute/chronic leukemia including AML, ALL, MDS, CML. 
Patients who are found to be in relapse only by molecular methods or cytogenetics will continue with 
the protocol therapy, except for cases which received conventional cytotoxic chemotherapy. The 
following FDA-approved tyrosine kinase inhibitors/hypomethylating agents are allowed in case of 
molecular/cytogenetic relapse: imatinib, nilotinib, dasatinib, ponatinib, sorafenib, azacitidine and 
decitabine. Participants who undergo disease relapse will cease all future study visits/procedures and 
will be followed only for survival through Day 365. 
 
5.3.6 
Clinical laboratory chemistry, hematology, EKG and pregnancy test 
A complete metabolic panel will include the following 18 blood chemistry parameters (CMP): glucose, 
BUN (blood urea nitrogen), creatinine, uric acid, total proteins, albumin, calcium, phosphorous, sodium, 
potassium, chlorine, total CO2, total bilirubin, alkaline phosphatase, ALT (alanine transaminase), AST 
(aspartate aminotransferase), LDH (lactate dehydrogenase), total cholesterol. 
An EKG will be performed at days 47 and 77 (+/- 10 days) to identify any vaccine related cardiac AEs if 
the patient is in a state of health that can tolerate the assessment. 
For women of child bearing potential, a serum or urine pregnancy test is acceptable. Female participants 
who are menopausal are not considered of child bearing potential. Menopausal status is defined as 
more than 12 months without menses, or a medical history of menopausal status (estrogen level and 
follicle stimulating hormone in the menopausal range).  
 
The hematology testing will include a complete blood count (CBC) with differential. 
 

 
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5.3.7 
Adverse event assessment (CTCAE) 
All adverse events will be assessed using NCI CTCAE v. 4.0, which can be found at the following link: 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf8.1. 
Adverse 
events recorded in the source documents and the case report forms include: 
o All events considered possibly, probably or definitely related to study agent till end of 
the study period (Day 365) 
o All grade 1 and 2 events that occur within 2 weeks from each vaccination 
o All grade 3/4/5 events will be reported from Day 28 through Day 100 
o All serious adverse events will be reported to the sponsor from Day 28 through Day 100 
o After Day 100, all deaths will only be reported in the follow-up visit page of the eCRF. 
5.3.8 
Physical exam, vital signs, medical history, baseline symptoms, and demographics 
Physical exam including a review of skin will be performed. Vital signs will include review of weight, 
heart rate, blood pressure, respiration rate, and temperature.  Height will only be required at baseline. 
Medical history is defined as any significant medical conditions up to Day 0, but prior to stem cell 
infusion.      Baseline symptoms are defined as any symptoms on Day 28 prior to vaccination. 
 
5.3.9 
Concomitant medications 
All medications, supportive care, blood products or radiation therapy taken or administered during the 
trial will be documented in the subject’s clinical/hospital record, using COH, MDA and Dana-Farber 
documentation guidelines.  Medications related to a serious adverse event will be collected in the study 
case report forms as appropriate:  
o Concomitant medications 
o Anti-viral medications, including indication, start and stop date 
o Immunosuppressive agents 
o Prednisone, or equivalent, dosage for the 7 days prior to vaccine administration 
o Prohibited medications 
5.3.10 Immunogenicity testing 
All participants will undergo serial blood sampling for future immunogenicity testing. Section 9.0 details 
regarding sample collection, storage, and processing procedures for immunogenicity studies.  
 
5.4 Criteria for Completing/Discontinuing Study Participation 
Participation may continue until one of the following criteria applies: 
o Participant does not meet criteria for Day 28 vaccine  (such participant are not 
randomized and will be replaced) 
o Completion of study procedures 
o Participant withdraws from the study 
o General or specific changes in the participant's condition that render the participant 
unacceptable for participation in the opinion of the treating investigator. 
Documentation of the reason for completing study participation and the date effective should be made 
in the medical record and appropriate eCRF.  The COH DCC should be promptly notified of the change in 
participant status. 

 
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5.5 Follow-Up and Duration of Participation 
The length and involvement of study participation will vary based on vaccine administration, disease 
relapse, or decision to withdraw from the study, as detailed in the subsections that follow:  
5.5.1 
Participants who do not receive Day 28 vaccine administration 
Participants who do not meet criteria for Day 28 vaccine will discontinue any further follow-up. 
5.5.2 
Participants who relapse after receiving vaccine administration 
Participants who relapse after receiving vaccine administration will have procedures performed and 
documented up to that time of determination of relapse, and then will be followed for survival only 
(including reason for mortality) until Day 365 post-HCT.   
5.5.3 
Participants who withdraw from the study after receiving a vaccine administration 
Participants who withdraw from the study after receiving a vaccine administration may continue with 
follow-up per the participant’s agreement: 
– 
may elect to continue study monitoring procedures without Day 56 vaccine (if not 
already administered),  
– 
may elect to continue to be monitored for survival (including reason for mortality) until 
Day 365 only, or  
– 
may elect to withdraw completely; further follow-up or assessments will not occur. 
5.5.4 
All other participants (vaccine administered, have not relapsed or withdrawn)  
All participants who received at least one vaccine, have yet to have disease relapse, and have not 
withdrawn from the study, will continue follow up assessments and research blood draws as indicated in 
the Study Activity Calendar (Section 10) through day 365 post HCT. Study participation will be completed 
on Day 365 Post-HCT.   
 
5.6 Supportive Care, Other Concomitant Therapy, Prohibited Medications 
In general, the use of any concomitant medication/therapies and supportive care deemed 
necessary/appropriate for the care of the participant are allowed, with the following exceptions: 
o No other investigational agents may be given to patients 
o Alemtuzumab or any equivalent in vivo T-cell depleting agent is not permitted in this study 
following HCT, because its administration results in in vivo depletion of B, T and dendritic 
cells, potentially negating any positive effect of vaccinating the recipient with CMV-MVA 
Triplex. 
o Preemptive therapy with CMV immunoglobulin or antivirals (GCV/VAL, FOS, Cidofovir, CMX-
001) is not allowed following HCT. GCV/VAL, FOS, Cidofovir, CMX-001 may be used according 
to institutional SOC for preemptive management of CMV viremia. In general, therapy should 
not commence until after CMV qPCR ≥500 gc/mL. For preemptive therapy when qPCR<500, 
the study PIs are to be consulted. 
o Prophylactic antiviral treatment for HSV, HHV6, EBV and adenovirus including the use of  
GCV/VAL, FOS, Cidofovir, CMX-001 may also suppress reactivation of CMV, thus will not be 
allowed in this study following HCT. Therapeutic use of these agents is permitted per 
institutional standard practice.. Acyclovir has no therapeutic efficacy against CMV and is 
allowed as standard of care to prevent HSV. 

 
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o Medications that might interfere with the evaluation of the investigational product are 
prohibited up to 14 days after the second vaccination (day 70 post-HCT). Medications in this 
category include, but are not limited to:  
• 
Live attenuated vaccines  
• 
Medically indicated subunit (Engerix-B for HBV; Gardasil for HPV) or killed vaccines 
(e.g. influenza, pneumococcal, or allergy treatment with antigen injections) 
O Steroid therapy with prednisone, or equivalent, is permitted (see Section 6.0 for specific 
steroid dose criteria for vaccinations). 
 

 
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6.0 
VACCINE ADMINISTRATION CRITERIA 
6.1 Day 28-Post-HCT Vaccine Administration Criteria 
In instances where the criteria are not clear cut, the treating clinician and both Site PIs will collaborate 
to make a determination. This consultation should be documented.  
Following randomization, a first injection (Day 28 vaccine) shall be administered if the following criteria 
are met on the day of vaccination: 
___ 15. 
NOT experienced ≥ Grade 3 GVHD within past 7 days 
___ 16. 
Disease has NOT relapsed since HCT 
___ 17. 
Successful primary engraftment WITHOUT secondary graft failure 
___ 18. 
NO ongoing post-HCT ≥ Grade 3 non-hem AE’s, with the exception of grade 3 
glucose intolerance, cholesterol, triglyceride, and hyperglycemia 
___ 19. 
Negative for CMV viraemia: CMV qPCR <500 gc/mL from samples collected and 
resulted within the past 7 days 
___ 20. 
Negative for CMV end organ disease (biopsy proven) post-HCT 
___ 21. 
All prednisone (or equivalent) doses within the past 7 days were  ≤ 1 mg/kg/day*  
___ 22. 
NOT received any prohibited medications (Section 5.6) 
___ 23. 
Negative pregnancy test result for females of child bearing potential 
The DCC should be promptly notified by the study team of registered participants who fail to meet Day-
28 Post-HCT vaccine administration criteria. 
 
6.2 Day 56-Post-HCT Vaccine Administration Criteria 
In instances where the criteria are not clear cut, the treating clinician and Site PIs will make a 
determination. This consultation should be documented.  
Day 56 vaccine shall be administered if the following criteria are met: 
___ 24. 
NOT experienced  Grade 4 GVHD since Day-28 vaccination 
___ 25. 
NO Grade 3 GVHD within the past 7 days 
___ 26. 
Disease has NOT relapsed since HCT 
___ 27. 
NO secondary graft failure 
___ 28. 
NO ongoing post-HCT ≥ Grade 3 non-hem AE’s, with the exception of grade 3 
glucose intolerance, cholesterol, triglyceride, and hyperglycemia 
___ 29. 
All prednisone doses (or equivalent) within the past 7 days were  ≤ 1.5 mg/kg/day* 
___ 30. 
NOT received any prohibited medications (Section 5.6) 
___ 31. 
Negative pregnancy test result for females of child bearing potential 
 
*Day 28 and day 56 vaccine administration criteria allow different doses of prednisone (or equivalent) 

 
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7.0 
DATA AND SAFETY MONITORING 
This study will have an external, independent DMC for review of protocol events and progress, with 
reporting of recommendations to the COH IRB.  
 
7.1 Definition of Risk Level 
This is a Risk Level 4 study, as defined in the City of Hope Data and Safety Monitoring Plan because the 
trial involves a COH held IND. 
 
7.2 Monitoring and Personnel Responsible (PMT) 
The PMT will consist of the study PI, site principle investigators, collaborating investigators, 
Biostatistician and CRA/protocol nurse. They will be responsible for monitoring the data and safety 
of this study, including implementing stopping rules for safety and efficacy. In addition, monitoring 
of research subjects following treatment will be conducted by the Clinical Trials Office and/ or 
designee. Study data and safety information from all participating sites will be collected at COH for 
consideration by the Independent DMC prior to DMC meetings (Meeting Schedule see Section 7.6). 
7.3 AE and UP Definitions 
7.3.1 
Adverse event (AE) 
An adverse event is any untoward medical experience or change of an existing condition that occurs 
during or after treatment, whether or not it is considered to be related to the protocol intervention.  
7.3.2 
Unexpected Adverse Event [21 CFR 312.32 (a)] 
An adverse event is unexpected if it is not listed in the investigator’s brochure and/or package insert; is 
not listed at the specificity or severity that has been observed; is not consistent with the risk information 
described in the protocol and/or consent; is not an expected natural progression of any underlying 
disease, disorder, condition, or predisposed risk factor of the research participant experiencing the 
adverse event. 
7.3.3 
Expected Adverse Event  
Any event that does not meet the criteria for an unexpected event OR is an expected natural 
progression of any underlying disease, disorder, condition, or predisposed risk factor of the research 
participant experiencing the adverse event. 
7.3.4 
Serious Adverse Event (SAE) [Modified from 21 CFR 312.32]  
Serious adverse events will be reported to the sponsor (COH) within 1 business day of study staff 
becoming aware of them. A serious adverse event is defined as any expected or unexpected adverse 
event up to Day 100 that results in any of the following outcomes: 
• 
Death 
• 
Life-threatening (places the subject at immediate risk of death from the event as it 
occurred)  
• 
Unplanned hospitalization (equal or greater than 24 hours) or prolongation of existing 
hospitalization 
• 
A persistent or significant disability/incapacity  
• 
A congenital anomaly/birth defect 

 
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• 
Secondary Malignancy 
• 
Any other adverse event that, based upon appropriate medical judgment, may jeopardize 
the subject’s health and may require medical or surgical intervention to prevent one of the 
outcomes listed above (examples of such events include allergic bronchospasm requiring 
intensive treatment in the emergency room or at home, blood dyscrasisas or convulsions 
that do not result in inpatient hospitalization, or the development of drug dependency or 
drug abuse). 
7.3.5 
Unanticipated problems Involving Risk to Subjects or Others  
An unanticipated problem is any incident, experience or outcome that meets all three of the following 
criteria: 
1. Unexpected (in terms of nature, severity, or frequency) given the following: a) the research 
procedures described in the protocol-related documents such as the IRB approved research 
protocol, informed consent document or Investigator Brochure (IB); and b) the characteristics of 
the subject population being studied; AND 
2. Related or possibly related to participation in the research (possibly related means there is a 
reasonable possibility that the incident, experience, or outcomes may have been caused by the 
drugs, devices or procedures involved in the research); AND 
3. Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm) than previously known or recognized. 
7.3.6 
AE Description and Grade 
The descriptions and grading scales found in the most recent version of Common Terminology Criteria 
for Adverse Events (CTCAE) version 4.0 will be utilized to characterize AEs, a copy of which can be found 
at http://evs.nci.nih.gov/ftp1/CTCAE/About.html. AEs not covered by specific terminology listed should be 
reported with common medical terminology, and documented according to the grading scales provided 
in the CTCAE Version 4. 
7.3.7 
AE Attribution 
The relationship of event to study drug will be documented by the Investigator as follows: 
• 
Unrelated: The event is clearly related to other factors such as the participant’s clinical state, other 
therapeutic interventions or concomitant drugs administered to the participant. 
• 
Unlikely: The event is doubtfully related to investigational agent(s). The event was most likely 
related to other factors such as the participant’s clinical state, other therapeutic interventions, or 
concomitant drugs. 
• 
Possible: The event follows a reasonable temporal sequence from the time of drug administration, 
but could have been produced by other factors such as the participant’s clinical state, other 
therapeutic interventions or concomitant drugs. 
• 
Probable: The event follows a reasonable temporal sequence from the time of drug administration, 
and follows a known response pattern to the study drug. The event cannot be reasonably explained 
by other factors such as the participant’s clinical state, therapeutic interventions or concomitant 
drugs. 
• 
Definite: The event follows a reasonable temporal sequence from the time of drug administration, 
follows a known response pattern to the study drug, cannot be reasonably explained by other 
factors such as the participant’s condition, therapeutic interventions or concomitant drugs; AND 
occurs immediately following study drug administration, improves upon stopping the drug, or 
reappears on re-exposure.  

 
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7.4 Routine Reporting of Adverse Events by Site Investigators 
Adverse events of all grades will be reported into the eCRFs by the study CRA. Adverse events recorded 
in the case report forms include: 
o All events, regardless of grade considered possibly, probably or definitely related to 
study agent 
o All grade 3/4/5 events regardless of attribution  
o All serious adverse events 
Information should include: participant ID, date of the event, whether the event meets the definition of 
serious, whether the event is an unanticipated problem, grade of event, attribution of event, whether 
the event is a known expected toxicity to study agent. Provide all possible causality to the event (e.g. 
subject’s disease, medical history, comorbidity(ies)) 
All adverse events must be followed until the event is resolved, stabilized, or determined to be 
irreversible by the participating investigator; for ongoing adverse events that are unrelated to study 
agent, the follow-up period may end at the 30-days post study-drug assessment. The Coordinating 
Center should be consulted prior to ending the follow-up of events that have stabilized. 
 
7.5 Expedited Reporting of Unanticipated Problems (UPs) by Site Investigators 
Each adverse event will be assessed to determine if it meets the criteria for expedited adverse event 
reporting.  UPs occurring at COH will be reported using the iRIS AE/UP reporting form according to the 
COH reporting timeline. 
Non COH sites will complete the UP/SAE Coversheet (found in Appendix E ) and email a scanned copy to 
the COH Data Coordinating Center with the subject title “CMV SAE” to DCC@coh.org as soon as possible 
(electronic signature on the document is acceptable). In addition, all UPs must be reported to the local 
IRB and copies of the IRB submission and response provided to the COH DCC.  All UP/SAE reports will be 
forwarded immediately to study Principal Investigator (rnakamura@coh.org). 
If an email receipt from Coordinating Center personnel is not received within one working day, external 
site personnel should call 626-256-4673 x 63968 and/or email DCC@COH.org. 
All UPs reported at COH and external sites will be compiled and submitted for review by the 
Independent DMC. 
 
7.5.1 
Rationale for Expedited Adverse Event Reporting to Local IRB 
The criteria defining expedited reporting is derived from FDA and COH reporting criteria. The following 
should be reported to the local IRB and Study PI within 24 hours of being aware that the event met the 
expedited reporting criteria: 
•  
All unanticipated problems 
• 
All serious adverse events regardless of relationship to study agent, study procedure, underlying 
disease or concomitant treatment from the first vaccine administration till 30 days after the vaccine 
dose. 
• 
All serious adverse events that are considered possibly, probably, or definitely related to the 
study agent observed 30 days after the last dose of vaccine 
 

 
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Note: follow-up reports must be submitted for all events that require expedited reporting when the 
status of the event changes and until the resolution or stabilization of the event. 
 
7.5.2 
Reporting of Unanticipated Problems to the FDA  
SAEs meeting the requirements for expedited reporting to the FDA, as defined in 21 CFR 312.32, will be 
reported by COH as an IND safety report using the MedWatch Form FDA 3500A for Mandatory 
Reporting which can found at: 
 http://www.fda.gov/Safety/MedWatch/HowToReport/DownloadForms/default.htm 
The COH PI or designee will be responsible for contacting the Office of IND Development and 
Regulatory Affairs (OIDRA) at COH to ensure prompt reporting of safety reports to the FDA.  OIDRA will 
assist the PI with the preparation of the report and submit the report to the FDA in accordance with the 
following: 
• 
any unexpected fatal or life threatening adverse experience associated with use of the drug 
must be reported to the FDA no later than 7 calendar days after initial receipt of the information 
[21 CFR 312.32(c)(2)]; 
• 
any adverse experience associated with use of the drug that is both serious and unexpected 
must be submitted no later than 15 calendar days after initial receipt of the information [21 CFR 
312.32(c)(1)] 
• 
any follow-up information to a study report shall be reported as soon as the relevant 
information becomes available. [21 CFR 312.32(d)(3)] 
 
7.6 Independent Data Monitoring Committee (DMC) 
Aggregate data (see Tables 1-5 of the DMC Charter) will be compiled by the Study Statistician. The COH 
DCC will report this study data to the independent Data Monitoring Committee. The Independent DMC 
will comprise three members with appropriate scientific and medical expertise to monitor the study 
progress (see Section 9.1 DMC Charter for Committee members). The DMC charter has been written by 
COH and agreed by all DMC members prior to the initial DMC meeting. A DMC chairperson will be 
appointed who will be responsible for conducting the meeting and summarizing the minutes of the 
closed portions of the meeting. All meetings will be held by teleconference, approximately a month 
after the following occurs:  
• 
approx 24 patients reach day 100 
• 
approx 48 patients reach day 100 
• 
approx 72 patients reach day 100 
• 
At study conclusion (optional final DMC meeting)  
After any NRM within 100 days, grade 3-4 aGVHD, grade 3-4 reaction with an attribution of ‘probably’ or 
‘definitely’  related to vaccination  (within 2 weeks of a vaccine administration), or any other concerning 
reaction as deemed by the PMT, the DMC chair will be provided information and will determine if a 
DMC review is required.  This information will be provided to the DMC chair as soon as practically 
possible after PMT review. 
NOTE. Modifications to this schedule may be required due to study progress/findings. 
 

 
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7.7 Toxicities to CMV-MVA Triplex 
7.7.1 
Expected (known) toxicities to CMV-MVA Triplex 
Expected (known) to be associated with CMV-MVA Triplex (in agreement with IB) in healthy volunteers 
and HCT recipients, with the highest grade indicated: 
Cutaneous reaction (grade 3), Myalgia (grade 1-2), Malaise (grade 1-2), Headache (grade 1-2) 
7.7.2 
Anticipated toxicities to CMV-MVA Triplex 
Anticipated toxicities that have not yet been seen from the agent but are foreseeable based on other 
similar agents include bruising at the site of injection and transient hypotension. 
All unsolicited AE data will be collected up to one month post vaccination. All SAE data will be collected 
throughout the duration of the study, for up to one year. 
 
7.8 Toxicities to the placebo  
7.8.1 
Expected (known) toxicities to placebo 
Since the placebo formulation has yet to be administered to participants, there are no expected (known) 
toxicities associated with administering the placebo.  
7.8.2 
Anticipated toxicities to placebo 
Anticipated toxicities that have not yet been seen from the placebo but are foreseeable based on other 
similar agents include bruising at the site of injection and transient hypotension. 
 
8.0 
AGENT INFORMATION 
8.1 CMV-MVA Triplex and placebo –information applicable to vaccine components 
CMV-MVA Triplex injection will be prepared by the investigational pharmacy from the following 
component provided by the COH CBG:  
 
cGMP CMV-MVA Triplex vaccine  vials containing 5.1x108  pfu/mL (Lot#0786-181-0001-1) OR 
9.1x108  pfu/mL (Lot#0786-181-0002-1). 
 
COH CBG has filled vials with cGMP CMV-MVA Triplex vaccine without lyophilization in the 
formulation buffer of PBS containing 7.5% lactose.  Vials contain approximately 1 mL. 
 
 
The placebo will be prepared by the investigational pharmacy from the following components provided 
by the COH CBG: 
• 
PBS containing 7.5% lactose.  
 
For all of the above listed components, the following elements apply: 
8.1.1 
Accountability 
The investigator, or a responsible party designated by the investigator, must maintain a careful record of 
the inventory and disposition of each of the agents using the NCI Drug Accountability Record or another 
comparable drug accountability form.  
In addition, documentation of the vaccine preparation (placebo or CMV-MVA Triplex) is required.  

 
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8.1.2 
Handling 
Qualified personnel, familiar with aseptic technique and procedures that ensure the quality of the agent 
and minimize undue exposure to themselves and the environment, should undertake the preparation, 
handling, and, when applicable, safe disposal of the agent.  
 
8.1.3 
Ordering 
The COH IDS pharmacy will order all agents from COH CBG in coordination with and as approved by Dr. 
Diamond (ddiamond@coh.org) and Dr. Nakamura (rnakamura@coh.org). 
The MDA pharmacy and Dana-Farber pharmacy will order all agents from the COH IDS pharmacy.  
8.1.4 
Destruction and Return 
Vials used in the preparation of vaccine for administration and any residual agents there in may be 
disposed of by the research pharmacy according to approved institutional standard of practice or policy. 
Unused agent will either be returned to the sponsor or disposed of according to approved institutional 
standard of practice. No unused agent will be disposed of without prior written approval by Dr. Don 
Diamond (ddiamond@coh.org).   
 
8.2 CMV-MVA Triplex  
8.2.1 
Description 
The CMV-MVA Triplex vaccine is a multiple-antigen recombinant MVA with genes encoding 3 major CMV 
proteins: UL83 (pp65), UL123 (IE1), and UL122 (IE2).  The CMV-MVA Triplex vaccine was manufactured 
at COH CBG, in a California Food and Drug Branch (CFDB) licensed manufacturing facility which operates 
under the principles of cGMP for the manufacture of phase I/II biologics.  The release testing of the 
vialed CMV-MVA Triplex vaccine to be used in the current Phase II trial was performed by BioReliance 
Corporation (Rockville, MA) and Wuxi-Apptec (Marietta, GA), in compliance with the requirements of 
the FDA Good Laboratory Practice Regulations (21 CFR 58). The vaccine passed all applicable release 
tests specified by the FDA. The toxicology testing of the cGMP CMV-MVA Triplex vaccine was performed 
at the Southern Research Institute (Birmingham, AL; Study # 13928.01.01). The IND is held by COH (BB-
IND #15792). 
8.2.2 
Toxicology 
Most common AEs included mild to moderate cutaneous reactions at the injection site. The duration of 
related Grade 1 and 2 AEs ranged from 1-2 days. One cutaneous grade 3 AEs (skin induration) was 
reported which was resolved in 2 days.  
8.2.3 
Supplier  
CMV-MVA Triplex vaccine is being produced, formulated and provided by COH CBG. COH IDS pharmacy 
or COH CBG will supply MDA and Dana-Farber with CMV-MVA Triplex vaccine for this study.  
CMV-MVA Triplex vaccine is supplied frozen at 5.1x108 pfu/mL or 9.1x108 pfu/mL in the formulation 
buffer of PBS containing 7.5% lactose.  COH CBG has filled vials with cGMP CMV-MVA Triplex vaccine 
without lyophilization as a sterile, preservative-free, solution packaged in 2 mL polypropylene cryogenic 
vials with silicone washer seals. Each vial contains approximately 1mL (0.9 - 1.1mL). 
8.2.4 
Storage and stability 
CMV-MVA Triplex is to be stored in a monitored freezer between -60 to -90 oC.  Stability analyses of 
CMV-MVA Triplex are performed every 6 months.  

 
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8.2.5 
Handling, Ordering, Accountability, Destruction and Return 
See section 8.1. 
8.3 PBS containing 7.5% lactose solution for the placebo 
8.3.1 
Description 
PBS containing 7.5% lactose is a sterile, nonpyrogenic solution which is the diluent used in the 
formulation of CMV-MVA Triplex. This solution will be used for the placebo injection.  
8.3.2 
Toxicology 
There are no known warnings. Lactose is a reducing sugar commonly used in multiple drugs as an 
excipient or bulking agent.  It is a natural disaccharide consisting of galactose and glucose [190]. It can 
be administered by different routes including IM, as in SOLU-MEDROL® (Pfizer, NY) the anti-
inflammatory glucocorticoid, often prescribed to HCT patients as in immunosuppressive agent. There 
are no restrictions for diabetes and lactose intolerant patients to take lactose containing medicines, 
since the amount of lactose delivered in drugs is minimal[190]. 
8.3.3 
Formulation 
PBS containing 7.5% lactose will be supplied in a 1.2 mL polypropylene cryovial  vial with a fill volume of 
approximately  1 mL/vial.  The solution contains no bacteriostat, antimicrobial agent or added buffer.  
8.3.4 
Supplier 
PBS containing 7.5% lactose is produced by COH CBG. The COH IDS pharmacy or COH CBG will supply 
PBS containing 7.5% lactose to MDA and Dana-Farber. 
8.3.5 
Storage and Stability 
PBS containing 7.5% lactose should be maintained between -60oC and -90oC in a temperature-monitored 
freezer. The release testing for PBS containing 7.5% lactose fill includes sterility, bacteriostasis and 
fungistasis, endotoxin, pH and particulate testing. The testing will be performed until the end of the 
study. 
8.3.6 
Handling, Availability, Ordering, Accountability, Destruction and Return 
See section 8.1. 
8.4 Preparation of CMV-MVA Triplex 
A list of reagents, equipment and supplies needed to prepare the vaccine is located in Appendix D. 
Note: the CMV-MVA Triplex vaccine must be administered within 4 hours after the CMV-MVA Triplex 
vaccine has thawed. 
Thawing and preparing CMV-MVA Triplex injection 
Before the scheduled patient administration, obtain frozen CMV-MVA Triplex vaccine vial from -70°C 
freezer in the Pharmacy.   
 
Put cooling block in hood and set to 4-8°C. Once the LCD on the cooling block reads 4-8°C, measure 
the temperature of the cooling block  using the NIST thermometer  and Enviro-Safe liquid or digital 
thermometer. Adjust LCD until  thermometer reads 4-8°C.  Only the  thermometer should be relied 
upon for accuracy. 
 
Allow vaccine vial to thaw at room temperature (approximately 15-30 minutes).  Record time at 
which vial is completely thawed. This is the start time of the vaccine dose preparation. Once 
thawed, place vial on the cooling block maintained at 4-8°C.  If there is any delay between thaw and 
the subsequent steps, keep the thawed vial on the cooling block until it is vortexed and spun. 

 
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 
 Thoroughly wipe the exterior of the vial(s) with an alcohol swab.  
 
Vortex vial for 30 seconds at highest setting (this is to minimize clumping). 
 
Spin for 5 seconds in a  microfuge at 6000 rpm (this is to maximize the extractable volume) .   
 
Unscrew the cap from the vaccine vial and withdraw CMV-MVA Triplex into an appropriately sized 
sterile syringe. If using Lot#0786-181-0001-1, withdraw the entire contents of vial (0.9-1mL of 
vaccine). If using Lot#0786-181-0002-1, withdraw 0.55 mL.  
 
Record the time that the CMV-MVA Triplex vaccine is placed in an insulated container with ice packs 
and is ready for transport. 
Labeling of the vaccine to communicate necessary information and maintain the blind 
 
The prepared syringe or accompanying documents should be labeled in a manner to maintain a 
blind randomization status with the clinical study team and participant. After labeling appropriately, 
place syringe in sealable, plastic amber bag and place in container with ice packs. 
 
The time by which the vaccine needs to be administered – start time +4 hours – must be clearly 
noted in order to inform the person administering the vaccine (e.g. “Administer before xx:xx 
am/pm”). 
 
8.5 Preparation of placebo 
Preparation 
Note: PBS containing 7.5% lactose solution for the placebo must be administered within 4 hours after 
the solution has been removed from the freezer. 
 
Put cooling block in hood and set to 4-8°C. Once the LCD on the cooling block reads 4-8°C, measure 
the temperature of the cooling block using the NIST thermometer and Enviro-Safe liquid or a digital 
thermometer. Adjust LCD until NIST thermometer reads 4-8°C.  Only the  thermometer should be 
relied upon for accuracy. 
• 
Obtain PBS containing 7.5% lactose solution from the Pharmacy freezer.  Record the time at which 
the PBS containing 7.5% lactose solution is removed from the freezer; this is the start time of the 
placebo dose preparation. Allow placebo vial to thaw at room temperature. Keep cold by placing 
vials on the cooling block, maintained at 4-8°C.   
• 
Vortex the vial (containing approximately 1 mL of volume) for 30 seconds at highest setting. 
Centrifugation is not required. 
• 
Withdraw 0.9-1 mL for the subject injection and cap syringe.  
• 
After labeling appropriately, place syringe in sealable plastic amber bag and place in ice bucket.  
Record the time that the placebo is placed in a container with ice packs and is ready for transport. 
 
 
Labeling of the vaccine to communicate necessary information and maintain the blind 
• 
The prepared syringe or accompanying documents should be labeled in a manner to maintain a 
blind randomization status with the clinical study team and participant. 
• 
The time by which the vaccine needs to be administered – start time +4 hours – must be clearly 
noted in order to inform the person administering the vaccine (e.g. “Administer before xx:xx 
am/pm”). 

 
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9.0 
CORRELATIVE/SPECIAL STUDIES 
9.1 Immunogenicity testing 
The correlative immunogenicity studies will include monitoring levels and quality of CMV-specific CD8+ T 
cells and highly cytotoxic memory NKG2C+ NK cells, by multi color flow cytometric analyses.  
9.1.1 
Specimen Collection and Transport To Processing Laboratory 
All participants will undergo serial blood sampling for immunogenicity testing at the schedule indicated 
in the Study Activity Calendar (Section 10), which, in general, includes days 28, 42, 56, 70, 84, 100, 140, 
180, 270 and 365 post-HCT. A volume of approximately 30 mL of blood in heparin (green-top) tubes, will 
be collected, gently inverted several times to mix anti-coagulant, and kept at room temperature until 
transport to authorized personnel of the DET at COH or at Dana-Farber or MDA.  Samples from all pre-
registered participants, including Pre-HCT samples from participants who are not randomized 
(vaccinated), will be maintained/included for analysis.   PLEASE SEE SECTION 9.2 
9.1.2 
Initial specimen processing, storage, and shipping 
Sample processing and storage will occur at COH, MDA or Dana-Farber. PBMC will be separated from 
heparinized blood by standard density gradient centrifugation methods*, washed, re-suspended in 90% 
fetal calf serum (FCS) with 10% DMSO, aliquoted, and cryopreserved in a centrally monitored liquid 
nitrogen tanks at the COH DET, MDA  and at the Dana-Farber Cancer Institute. Samples will be 
processed and stored according to institutional SOP; it is recommended to freeze 5 million cells per 
aliquot.  Samples will be labeled with the study timepoint (e.g. Day 42), date of collection, and study 
protocol number (COH 14295) , participant study number and site (e.g. MDA, COH or Dana-Farber).   
* Prior to the processing of the PBMCs, 3 mL of blood  will be set aside for plasma extraction. This 3mLs 
of blood will be centrifuged at around 3,000g for 20 mins to spin down the cell fraction. After spinning, 
the upper layer of plasma is removed , transferred to labeled cryotubes  and stored at in a suitable 
laboratory freezer between -20oC and -70C, or liquid nitrogen. The cell fraction will be discarded. When 
requested by the ET Laboratory who are performing the analysis, frozen samples will be batch shipped 
from external sites, overnight on dry ice to COH. 
 
9.1.3 
Analytical Method 
CMV-specific CD8 and CD4  T cells  
Immunogenicity studies of CMV-specific T cells will be performed at the COH DET and will include 
immune-monitoring of levels, kinetics, poly functionality and phenotype assessment of CMV-specific T 
cells[15, 20, 28]. Additionally, CMV-specific T cell growth kinetics and early cell death we be monitored. 
The proposed Phase II blinded placebo controlled study will definitively assess the level of association 
between reduced CMV reactivation and CMV specific T cells. 
Multicolor FACS analyses will be used to assess poly functionality and phenotype of CMV-specific T 
cells[15, 20, 28]. All immune-monitoring studies will be performed by flow cytometry techniques using 
the Gallios TM with Kaluza software (Beckman Coulter Inc, Brea CA). 
We will evaluate the cellular CMV-specific response elicited by CMV-MVA Triplex by measuring the 
levels of the CD137 surface marker expressed on CD8 and CD4 T cells stimulated for 24 hours with either 
pp65, IE-1 and IE2 peptide libraries[182]. CD137 is expressed only on recently activated T cells, and its 
expression correlates with functional activation of T cells[183, 184]. All immunophenotyping will be 
conducted on freshly thawed PBMC without cultivation or stimulation in vitro.  PBMC will be stained 
with each fluorochrome-conjugated antibody combination using standard methods with commercially 
fluoresceinated antibodies (BD Biosciences, San Jose, CA), as described in our published studies[140, 

 
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191].  In combination with CBC, we will be able to calculate the absolute number of pp65/IE1/IE2–
specific CD8 and CD4 T cells/L.   
Assessment of CMV-specific T cell growth kinetics will be performed using the carboxyfluorescein 
diacetate succinimidyl ester (CFSE) dilution method for cell division tracking as previously detailed[182]. 
Briefly, proliferation will be analyzed using the CFSE dilution method, according to the manufacturer’s 
procedure (Molecular Probes, Carlsbad, CA). To assess CD8 T cell proliferation, PBMC will CFSE labeled 
and incubated for 6 days with 1 µg/mL pp65495-503 peptide or DMSO  diluent as control, in the presence 
of anti-CD49b and CD28 (1 µg/mL). Cells will be then washed and co-stained with anti-CD8 (all 
antibodies from BD Biosciences) and FACS analyzed for CFSE fluorescence levels. Early cell death will be 
determined using the BD ApoAlert annexin V-FITC Apoptosis Kit (BD Biosciences) according to the 
manufacturer’s instructions, and cells will be analyzed by  FACS [192]. 
Natural Killer cell phenotype and function 
Assessment of NK phenotype and function (cytotoxicity and cytokine production) will be performed at 
UMN. These studies will determine whether vaccination of  allogeneic HCT-R+ induces adaptive NK cell 
population changes, and increase in the highly cytotoxic memory NKG2C+ NK cells, linked to CMV 
reactivation, critical for CMV adaptive immune response and potentially linked to relapse reduction[24-
27]. Since in the pilot Phase Ib appeared to reduce reactivation, cGVHD and relapse we will assess 
whether vaccinated patients have increased levels of this potent subset of NK cells[25-27]. The NK 
analyses will be performed at a single cell level by using a 9-color flow approach[25]. 
 GVHD Biomarkers 
In the phase Ib trial of CMVPepVax in HCT recipients (COH IRB Clinical Protocol No. 12022), vaccine 
administration was associated with reduced risk of chronic GVHD while there was no increase in acute 
GVHD. To further investigate potential immunologic impacts of CMV-MVA Triplex on GVHD, the 
following GVHD biomarkers will be evaluated at the COH DET: 
o interleukin-2 receptor α (IL2Ra), tumor necrosis factor receptor 1 (TNFR1), hepatocyte 
growth factor (HGF), interleukin 6 (IL6) and interleukin-8 (IL8) for systemic GVHD[171, 172] 
o elafin for skin GVHD[173] 
o regenerating islet-derived 3α (REG3α) for gastrointestinal GVHD[174] 
o suppression of tumorigenicity 2 (ST2) for steroid-refractoriness [175] 
o CXCL9 and B cell–activating factor (BAFF) for chronic GVHD[176, 177] 
The levels of these GVHD biomarkers will be compared between the vaccine group and placebo group, 
as well as between GVHD+ and – groups. 
9.2 MVA vector persistence 
As recommended by FDA, persistence of the CMV-MVA Triplex vaccine in the blood of vaccinated 
patients will monitored during the 12 month observation period after receipt of both doses of vaccine. 
At day 28, 56, 100, 180 and 365 one additional  3 mL  blood sample  with   anti-coagulant  will be 
collected for this evaluation. The method in use is detailed in paragraph 2.4.2.1. Samples collected at 
external trial sites will be frozen as whole blood, or cell fractions prior to batch shipping to COH for 
analysis. If samples are collected in non glass vacutainer tubes, they can be frozen in the collection tube 
with no further processing. Whole blood or cell fractions should be frozen and stored in a suitable 
laboratory freezer between -20oC and -80oC until shipping to COH. DNA will be extracted from the white 
cell fraction of the blood and qPCR analysis performed. The analysis will be performed in 1 single batch 
at end of the study after un-blinding procedures have been completed.  

 
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10.0 STUDY CALENDAR 
Table 2: Study Activity Calendar  
 
Pre-HCT 
HCT Day 0 
 
 
Vaccination and Bi-
Weekly Visits b 
Post-100 Day 
Follow-Up c 
Study day a 
**(-60 - 0) 
28d 42 47 56d 70e 77 84 100 140 180 270 365 
Informed Consent f 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history and 
demographics g 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Concurrent medications h 
X i 
X j 
X j 
 
X j X j 
 
X j X j 
X k X k 
X k X k 
Physical exam and vital signs l 
X 
X 
 
 
X 
 
 
 
 
 
 
 
 
KPS performance status m 
X 
X 
 
 
X 
 
 
 
 
 
 
 
 
Adverse event (CTCAE) assessment 
n
 
X 
X 
 
X 
X 
 
X 
X 
 
 
 
 
Engraftment status o 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
Disease relapse p 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
GVHD assessment and grading q 
 
X r 
X 
 
X s X 
 
X 
X 
X 
X 
X 
X 
CMV disease t 
 
X u 
 
 
X u 
 
 
 
 
 
 
 
X v 
CMV qPCR w 
 
X 
X 
 
X 
X 
 
X 
X -------------Xx-----------
HIV, HCV, CMV, active HBV y 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test z 
X 
X 
 
 
X 
 
 
 
 
 
 
 
 
Chemistry/metabolic panel aa 
X 
X 
 
 
X 
 
 
 
 
 
 
 
 
CBC with differential bb 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
Research blood sample cc 
 
X* 
X 
 
X* 
X 
 
X 
X* 
X 
X* 
X 
X* 
Criteria review 
X dd 
X ee 
 
 
X ff 
 
 
 
 
 
 
 
 
Registration/verify with DCC 
X gg 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccine administration hh 
 
Xhh 
 
 
Xhh 
 
 
 
 
 
 
 
 
EKG 
 
 
 
 
Xii 
 
 
Xii 
 
 
 
 
 
 
** Criteria review, screening, consent and Registration with DCC permitted Day -60 to Day 0 
a. Study day is defined relative to the day of HCT which is defined as Day 0. 
b. Window for post-HCT Day 28 and Bi-Weekly Assessment procedures is the assigned day +/- 5 days.  
c. Windows for the ‘Post-100 Day Follow-Up’ visits is the assigned day +/- 15 days. 
d. All assessments to be performed and reviewed on the day of and prior to vaccine administration, 
except for CMV qPCR for which the result may remain pending.  
e. On Day 70 post-HCT, 14 days after the second vaccination, administration of other vaccines is no 
longer prohibited (Section 5.6). 

 
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f. 
Informed consent process to be fully documented: e.g. prospective participant had sufficient time 
for deliberation, all questions were answered, treatment options provided by MD, full study 
reviewed including risks, and a copy of signed consent given to participant. Informed consent must 
occur prior to any research only (non-SOC) screening procedures. 
g. Medical history and demographics– to include any ongoing medical conditions and medical history 
pertaining to eligibility on study and involvement during study.  
h. Concurrent medications, supportive care, blood products, or radiation therapy taken or 
administered during the trial will be documented in the subject’s medical record using institutional 
documentation guidelines. 
i. 
Concurrent medications pertaining to eligibility criteria will be reviewed. 
j. 
Concurrent medication data collected in CRFs pertains to anti-viral medications (including start and 
stop date), immunosuppressive agents, daily prednisone dose for 7 days prior to vaccine 
administration, and prohibited medications (Section 5.6).   
k. Concurrent medication data collected after 100 days is limited to anti-viral medications (including 
start and stop date). 
l. 
Physical exam to include skin assessment. Vital signs:  Weight, heart rate, blood pressure, 
respiration rate, temp.  Height required only at baseline. 
m. KPS scale is found in Appendix C.  
n.  Adverse events (AEs) will be assessed documented in the source documents and the case report 
forms in the defined study visits and at study of care visits. All grade 1 and 2 AEs that occur within 2 
weeks from each vaccination will be reported. All grade 3/4/5 AEs will be reported from Day 28 to 
Day 100. All serious adverse events as defined in Section 7.3.4 will be reported up to Day 100. All 
AEs considered possibly, probably or definitely related to study agent will be reported till the end of 
the study period (Day 365).  See Section 7.0 for AE reporting. 
o. Engraftment status should be documented at each study visit, and if engraftment failure occurs the 
date of engraftment failure should be noted (see Section 5.3.3 for definitions).  
p.   See Section 5.3.4 for definition of disease relapse. Disease relapse will be assessed according to 
and per the timing of institutional SOC practice for the participant’s specific hematologic 
malignancy. Note: participants who undergo disease relapse will cease all future study 
visits/procedures and will be followed only for survival through Day 365. 
q. Acute and chronic GVHD grading scales are found in Appendix A and B, respectively. The final 
grading may occur after a clinic visit has ended, using all diagnostic information available to 
determine the GVHD grade at the time of the visit. In the study CRF, the grade at the time of the 
study visit will be recorded, and the highest grade and date of onset during the interim period 
between visits (assessed at SOC visits), if higher than the grade at the visit. 
r. For the Day 28 visit, assessment and grading of GVHD for the 7 days prior to the visit must be 
determined and documented on the day of the visit to assess/support vaccine administration 
criteria. The determination may be revised as more information is available, but will be 
documented in the medical record and in the CRF as a revised GVHD assessment and grading 
following new information obtained after vaccine administration. 
s. For the Day 56 visit, assessment and grading of GVHD between the initial vaccination and Day 56 
must be determined and documented on the day of the visit to assess/support vaccine 
administration criteria. The determination may be revised as more information is available, but will 
be documented in the medical record and in the CRF as a revised GVHD assessment and grading 
following new information obtained after vaccine administration. 

 
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t. Clinically confirmed CMV disease will be captured in the case report forms. Presentations or 
suspected presentations of CMV disease in the absence of qPCR >500 gc/mL will be evaluated by 
the treating investigator in conjunction with the blinded PMT before a determination is made.  
u. Participants with suspected CMV disease on the day of planned vaccination must have testing to 
confirm the presence or absence of CMV disease prior to determining for the CMV disease 
vaccination administration criterion.  
v. For the Day-365 visit, the treating investigator will investigate and document whether CMV disease 
occurred Day 100 onward, and so note the findings accordingly.  The case report forms will be 
updated to record any positive results not yet documented. 
w. CMV qPCR will be performed per institutional SOC (usually twice weekly), and at minimum weekly 
between days 21 and day 100 post-HCT. CMV results will be collected in the case report forms.   
x. Post Day 100, CMV qPCR to be performed only if clinically indicated or per institutional SOC. All 
CMV qPCR results will be collected in the case file.  
y. CMV serostatus, HIV antibody, Hepatits C antibody, Hepatitis B surface antigen test must be 
performed if results are not available within 2 months of registration. If CMV seropositive status 
not already documented, CMV serotesting may be performed for eligible review. 
z. Serum or urine pregnancy test to be performed for women of child bearing potential. 
aa. Chemistry/metabolic panel to include: glucose, BUN, creatinine, uric acid, total proteins, albumin, 
calcium, phosphorous, sodium, potassium, chlorine, total CO2, total bilirubin, alkaline phosphatase, 
ALT, AST, LDH, total cholesterol. 
bb. CBC with differential is to be taken at the same time as the research blood sample.  
cc. Research blood samples are to be collected at the same time as the CBC with differential. A volume 
of approximately 30 mL of Blood will be collected in heparin (green-top) tubes, gently inverted 
several times to mix anti-coagulant, and then kept at room temperature until transport to 
authorized personnel of the COH DET, MDA or Dana-Farber Research laboratory. Approximately 27 
ml and 3 ml are allocated for PBMC and plasma isolation respectively. * indicates that at Day 28, 
56, 100, 180, 365 one additional tube with approx 3 mL of blood   will be collected for the 
evaluation of the persistence of the MVA vector (see Section 9.2). In addition, in the case of a 
detectable CMV reactivation, external study sites will collect an extra tube (5mls, EDTA) at each 
research blood time point until day 100, for CMV harmonization measurements. 
dd. Eligibility criteria for enrollment in the clinical trial are found in Section 3.0.  
ee.  Day-28 Vaccine Administration Criteria are found in Section 6.1. Participants failing to meet Day-28 
vaccine criteria will complete the study at this time and be replaced and the study team should 
promptly inform the DCC.  
ff. Day-56 Vaccine Administration Criteria are found in Section 6.2. Participants failing to meet Day-56 
vaccine criteria will continue with remaining study procedures; participants with disease relapse 
will continue for survival follow-up only.  
gg. The study team will submit copies of source documentation, the eligibility criteria list, signed 
consent, and registration cover sheet to the DCC who will proceed to confirm study eligibility and 
register the participant.  See Section 4.2 for details.  
hh. Vaccine administration is detailed in Section 5.0. 
ii. Monitoring for cardiac AEs by EKG can be performed +/- 10 days of the assigned visit. 
 

 
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11.0 ENDPOINT EVALUATION CRITERIA/MEASUREMENT OF EFFECT 
The primary aims of this trial are to estimate the efficacy of CMV-MVA Triplex in reducing the frequency 
of CMV events, and to further evaluate the safety and tolerability in HCT recipients. Secondary 
endpoints will include additional safety, clinical and immunological parameters. In detail: 
 
Primary endpoints:  
1. CMV events encompassing any CMV reactivation [≥500 CMV genome copies (gc)/mL], low-level 
reactivation prompting antiviral therapy, or CMV disease prior to day 100 post-HCT. 
2. Key safety endpoints: non-relapse mortality (NRM) at 100 days post HCT, severe (grade 3-4) 
acute GVHD (aGVHD), and grade 3-4 AEs (CTCAE v.4.0) probably or definitely related to the 
vaccination within 2 weeks from each vaccination. 
 
Secondary endpoints:  
1) CMV-related events:  time-to viremia (defined as number of days from transplantation to the 
date of >500 CMV gc/mL), duration of viremia, incidence of late CMV viremia (>100 and ≤ 360 
days post HCT), use of antiviral drugs (triggered by clinically significant viremia of ≥1500 CMV 
gc/mL), cumulative number of CMV specific antiviral treatment days. 
2) Transplant-related events: time to engraftment, incidence of aGVHD, chronic GVHD (cGVHD), 
relapse,   non-relapse mortality, all-cause mortality, infections.   
3) Immunological function:  levels and kinetics of CMV-specific T cell immunity, combined with 
immunophenotyping[19-21], and functional studies[22, 23]. NK phenotype and function 
(cytotoxicity and cytokine production). 
 

 
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12.0 STATISTICAL CONSIDERATIONS 
12.1 Study Design 
This is a randomized, blinded and placebo controlled Phase II trial conducted at three centers, COH, 
MDA  and Dana-Farber with data coordination at COH.  The primary aims of the trial are to assess the 
ability of CMV-MVA Triplex to protect CMV-positive HCT recipients (HCT R+) from CMV reactivation 
events, as defined in Section 11, and to evaluate safety.  A secondary aim is to estimate the effect of 
CMV-MVA Triplex on levels of CMV cellular immunity in HCT-R+[161-163] and to describe the 
association of CMV cellular immunity with CMV reactivation events.  Additional secondary aims are to 
evaluate the effect of CMV-MVA Triplex on other transplant-related outcomes and on the duration and 
treatment of viremia.  The study includes rules for periodic safety monitoring. 
12.2 Randomization 
The trial has a target of 102 randomized HCT recipients.  Eligible HCT-R+ will be consented and enrolled 
pre-HCT, and registered through COH DCC.  A computer-generated randomization, stratified by donor 
CMV serostatus and center, will assign registered participants to the CMV-MVA Triplex or placebo arm; 
the treatment assignment will be provided to site pharmacists, who are unmasked to treatment-group 
allocation, in advance of planned vaccination. Registered participants will be followed for the course of 
transplant and be assessed for the eligibility for ‘Day-28 post-HCT’ vaccination. 
Participants who meet the initial vaccine administration criteria will receive the initial injection (vaccine 
or placebo); participants failing to meet the criteria will be replaced.  Only participants who receive an 
injection (CMV-MVA Triplex or placebo) will be considered “randomized”, as there is no opportunity for 
a drop-out bias prior to injection in this double-masked study.  All participants who receive a first 
injection will be included in the primary analysis, regardless of receipt of the second injection, consistent 
with the intention-to-treat principle.  Information regarding registered participants who do not receive 
the planned injection will be entered into the computer-generated randomization log to inform 
subsequent treatment assignments (their assignments will be replaced at randomly designated slots in 
the randomization log). Participants who do not meet the criteria to receive the first injection (CMV-
MVA Triplex or placebo) will leave the study, without further treatment or follow-up, but their data 
collected up to study departure will be included in the trial dataset as non-randomized subjects, and 
these data will be incorporated into estimates of CMV event rates post-HCT based on the intention to 
treat principle. 
12.3 Sample Size Accrual Rate 
A 40% rate of viremia (CMV reactivation) is expected among unvaccinated, eligible HCT recipients, with 
some possibility of a rate as low as 30%.  Based on the aim to detect either a vaccine-effect resulting in a 
drop from 40% to 15% or a drop from 30% to 10%, we plan to enroll and inject 51 subjects in each 
group.  The sample size will provide at least 90% power under either scenario, at a one-sided 0.10 level 
of significance (appropriate to a phase II trial, and conservatively estimated based on binomial 
incidence). The sample will also allow at least 83% power to achieve a 0.05 level of significance.  
Allowing for 10%-20% of subjects becoming ineligible before vaccination, we expect to enroll 
approximately 114-130 subjects in 2 years, to vaccinate 102-104, and observe between 20 and 28 
reactivation events.  The 102-104 HCT-R+ will be randomized, at or near day 28, in an approximate 1:1 
ratio, to either the CMV-MVA Triplex vaccine arm, or to the placebo arm.  Over the past five years, COH, 
MDA and Dana-Farber combined have performed on-average >300 adult allogeneic HCT procedures 
annually. We anticipate about 150 CMV seropositives to be eligible annually. Accrual should be 
completed in <4 years from the start date of the trial, and we anticipate 1 year of follow up and data 
analysis.  

 
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12.4 Data Analysis Plan 
CMV Events  
For the primary efficacy aim, each randomized study subject will be followed according to the study 
calendar (see Study Calendar, section 10.0) for the occurrence of CMV reactivation events, which are 
defined in section 11 in terms of either viremia, low-level viremia treated with antivirals, or CMV 
disease.  Both initial and recurrent events will be recorded, with patients considered at risk for recurrent 
events after completion of a full planned course of anti-viral therapy.  If PCR results from blood drawn 
on the day of first injection should indicate reactivation, the subject will be replaced and excluded from 
the primary comparison, but the subject will, in all other respects, continue on the study with all 
planned treatments and data collections. Vaccine and placebo groups will be compared with regard to 
the hazard of CMV events, using the Anderson-Gill approach to repeated events, as implemented in the 
R survival package, as we have previously published.  Power calculations are conservatively based only 
on binomial incident event rates. 
 
Safety 
The primary safety aim will involve both safety monitoring of the vaccine arm, described below in 
section 12.5, and comparison of arms at the end of the study.  Non-relapse mortality at 100 days will be 
summarized by arm as percentages, with exact binomial confidence bounds, and compared across arms 
using Fishers exact test.  The incidence of severe acute GVHD will be summarized by Kaplan-Meier 
curves, and compared using the log-rank test.  Any grade 3-4 SAE within two weeks of injection that are 
at least possibly attributed to the injection by the (masked) treating physician, will be listed, with 
incidence compared by Fisher’s exact test.  
 
Cellular immunity 
The data analysis for the secondary aim of estimating the effect of vaccination on cellular immunity will 
necessarily be more exploratory in nature.  The longitudinal CMV-specific cellular assay data will be 
modeled on a logarithmic scale, using a generalized estimating equation approach to accommodate the 
stochastic dependence through time.  This produces an estimated multiplicative effect of vaccination, 
qualified by a valid estimate of variability. 
 
Viremia duration and treatment 
The total days on antivirals for CMV reactivation (induction, maintenance, and total) will be assessed for 
each individual, with arms compared using Wilcoson’s rank-sum test.  Other characterizations of the 
duration of viremia and response to therapy will be descriptive in nature. 
 
Transplant-related events 
The cumulative incidence of acute GVHD, chronic GVHD, relapse-free survival, and infections will be 
estimated using Kaplan-Meier or Fine-Gray estimators, as appropriate, and compared using the log-rank 
test, or the corresponding subdistribution test due to Gray.  Computing will be dose using the survival 
and cmprsk R packages.  
 

 
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12.5 Safety Monitoring 
Clinical data will be monitored as they accumulate, and vaccination will be suspended for safety review 
if there is evidence of serious treatment-related AEs. Specifically: 
(1) 100 days non-relapse mortality (NRM) will be monitored as the 12th, 24th and 36th subject on 
the vaccine arm reaches the 100 day evaluation point. Operationally, the CRA will notify the 
monitoring statistician as cohorts of 24 patients (12 vaccinated) near the 100 day mark. If NRM 
frequencies exceed 4, 6, or 8 patients, at the designated 100 day evaluation point, then the trial 
will be suspended for DSMB safety review. These numbers were selected to limit the overall 
false-alarm probability for this endpoint to less than 0.02 when there is no additional risk due to 
immunization. 
(2) Severe acute GVHD (aGVHD, grade 3-4) will be monitored as the 12th, 24th, and 36th subject on 
the vaccine arm reaches the 100 day evaluation point. The trial will be interrupted if 6 or more 
of the first 12 recipients, or 9 of 24, or 11 of 36, experience Grade 3-4 aGVHD. This would be a 
significant elevation from the COH/MDA/Dana-Farber historical benchmark of 15% of allogeneic 
HCT recipients with matched sibling donors [30].  These rules are determined to limit the overall 
aGVHD false alarm probability to 0.02 if vaccination does not increase risk[30].  
(3) Serious AEs (SAE, grade 3-4) probably or possibly related to the vaccine (within 2 weeks from 
each vaccination) will be individually reviewed by the protocol monitoring team, and reported to 
the DSMC. 
 
Interim monitoring involves judging the vaccine arm against fixed benchmarks, to maximize power early 
in the study.  Comparison of treatment groups with respect to safety endpoints will occur at the end of 
the trial, and will use standard time-to-event statistical methods. 
 
 

 
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13.0 DATA HANDLING, DATA MANAGEMENT, RECORD KEEPING 
13.1 Source Documents 
Source documents are original documents, data, and records (e.g., medical records, pharmacy 
dispensing records, recorded data from automated instruments, laboratory data) that are relevant to 
the clinical trial.  The Site Investigator or their designee will prepare and maintain adequate and 
accurate source documents.  These documents are designed to record all observations and other 
pertinent data for each patient enrolled in this clinical trial.  Source documents must be adequate to 
reconstruct all data transcribed onto the case report forms. 
13.2 Data Capture Methods and Management 
Data for this trial will be collected using Medidata RAVE, City of Hope’s electronic capture system. 
Medidata RAVE is a web based, password protected system that is fully compliant with global regulatory 
requirements, including 21CRF Part 11 compliant.  
Study personnel at each site will enter data from source documents corresponding to a subject’s visit 
into the protocol-specific electronic Case Report Form (eCRF).  A system of computerized data validation 
checks will be implemented and applied to the database on a regular basis. Queries are entered, 
tracked, and resolved through the EDC system directly. The study database will be updated in 
accordance with the resolved queries.  All changes to the study database will be documented. 
The Data Coordinating Center will run monthly data expectation reports that will list any outstanding 
and overdue data. The Data Coordinating Center will send via email to the participating site a report 
monthly on any missing and/or overdue data forms.  The participating site will be required to complete 
the missing and/or overdue data forms within 1 week of receipt of the report. 
Query reports will be generated on a monthly basis by the Data Coordinating Center. The Data 
Coordinating Center will send via email to the participating site a report monthly on any outstanding 
queries.   
The participating site staff (whether Principal Investigator or the staff collecting data at site) are 
required to take an eLearning Module within Medidata RAVE in order to obtain full access. The 
participating site staff will receive training via teleconference by COH DCC staff to review eCRFs that are 
specific to this protocol.  Continuous training will be offered to participating sites if any amendments 
affect changes to the eCRFs during the course of the trial. The eCRFs within Medidata RAVE for this trial 
will have detailed instructions in the form of Help Text that provide instructions for completing each 
required field on each form. 
 
13.3 Case Report Forms/Data Submission Schedule 
Study personnel at each site will enter data from source documents corresponding to a subject’s visit 
into the protocol-specific electronic Case Report Form (eCRF) when the information corresponding to 
that visit is available.  
The Investigator is responsible for all information collected on subjects enrolled in this study.  All data 
collected during the course of this study must be reviewed and verified for completeness and accuracy 
by the Investigator.  All case report forms must be completed by designated study personnel.  The 
completed case report forms must be reviewed, signed and dated by the Site Investigator or designee in 
a timely fashion. 
All data will be collected using electronic data collection system described in Section 13.2, and will be 
submitted according to the timelines indicated in Table 2 (below). 

 
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Form 
Submission Timeline 
Eligibility Checklist 
Complete prior to registration  
On Study Forms 
Within 14 calendar days of registration 
Baseline Assessment Forms 
Within 14 calendar days of registration 
Treatment Forms 
Within 14 calendar days of treatment administration 
Adverse Event Report Forms 
Within 14 calendar days of the study visit 
Response Assessment Forms 
Within 10 calendar days of the response assessment 
Other Assessment Forms (concomitant 
medications, chemistry, hematology etc.) 
Within 10 calendar days of the assessment 
Off Treatment/Off Study Forms 
Within 10 calendar days of completing treatment or being 
taken off study for any reason 
Table 2: Data Submission Schedule 
 
13.4 Regulatory Records 
The Investigator will maintain records, including updating records in accordance with Good Clinical 
Practice guidelines and FDA regulations. Additional information regarding required documents is 
provided in the DCC Operations Manual, a supplement to this protocol. 
 
 

 
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14.0 ADHERENCE TO THE PROTOCOL 
It is understood that deviations from the protocol should be avoided, except when necessary to 
eliminate an immediate hazard to a research participant. Protocol deviations may be on the part of the 
subject, the investigator, or study staff.  As a result of deviations, corrective actions are to be developed 
by the study staff and implemented promptly. 
All deviations from the protocol must be documented in study subject source documents and promptly 
reported to the Study PI.  Protocol deviations will be submitted according to study site procedures to the 
local IRB (e.g. IRIS at COH), but programmatically exempted from local DMC review. A deviation report, 
which will include protocol deviations, will be prepared every 3 months for consideration by the COH 
PMT and Independent DMC.   
14.1.1 Emergency Modifications 
Investigators may implement a deviation from the protocol to eliminate an immediate hazard(s) for the 
protection, safety, and well-being of the study patient to trial subjects without prior IRB or Sponsor 
approval.  
For any such emergency modification implemented,  
o the local IRB must be notified according to local institutional policies.  
o the Study Principal Investigator must be notified as soon as practicable (within 24 hours) via 
email to rnakamura@coh.org and dcc@coh.org.  This email should provide input on the 
following: 
– 
Description of the event 
– 
Impact on participant safety or the safety to others 
– 
Impact on the study design 
14.1.2 Planned Non-Emergency Deviations 
All non-emergency planned deviations from the protocol must have prior approval by the Study 
Principal Investigator, the Site Principal Investigator and the local IRB, and if applicable the COH IRB. 
 Unplanned Deviations – Deviations Discovered After They Have Occurred 
For deviations to the protocol discovered after they have occurred,  
o the local IRB must be notified according to local institutional policies.  
o the Study Principal Investigator must be notified as soon as practicable (within 24 hours of 
awareness of event) via email to rnakamura@coh.org and dcc@coh.org.  This email should 
provide input on the following: 
– 
Description of the event 
– 
Impact on participant safety or the safety to others 
– 
Impact on the study design 
– 
A corrective and preventative action plan 
A list of deviations from all participating sites will be submitted along with the PMT progress report to 
the external DMC. 
 
 

 
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15.0 STUDY OVERSIGHT, QUALITY ASSURANCE, AND DATA & SAFETY MONITORING 
15.1 Site Principal Investigator  
The Principal Investigator is responsible for the conduct of the clinical trial at the site in accordance with 
Title 21 of the Code of Federal Regulations. The Principal Investigator must assure that all study site 
personnel, including sub-investigators and other study staff members, adhere to the study protocol and 
all FDA/GCP/NCI regulations and guidelines regarding clinical trials both during and after study 
completion. 
An investigator is responsible for ensuring that an investigation is conducted according to the signed 
investigator statement, the investigational plan, and applicable regulations; for protecting the rights, 
safety, and welfare of subjects under the investigator's care; and for the control of drugs under 
investigation.  
The Investigator agrees to: Conduct the study in accordance with the protocol and only make changes 
after notifying the Sponsor (or designee), except when necessary to protect the safety, rights or welfare 
of subjects. Personally conduct or supervise the study (or investigation). Ensure that the requirements 
relating to obtaining informed consent and IRB review and approval meet federal guidelines, as stated in 
§ 21 CFR, parts 50 and 56. Report to the Sponsor or designee any AEs that occur in the course of the 
study, in accordance with §21 CFR 312.64. Ensure that all associates, colleagues and employees assisting 
in the conduct of the study are informed about their obligations in meeting the above commitments. 
Maintain adequate and accurate records in accordance with §21 CFR 312.62 and to make those records 
available for inspection with the Sponsor (or designee). Ensure that an IRB that complies with the 
requirements of §21 CFR part 56 will be responsible for initial and continuing review and approval of the 
clinical study. Promptly report to the IRB and the Sponsor (see Section 14) all changes in the research 
activity and all unanticipated problems involving risks to subjects or others (to include amendments and 
IND safety reports). Seek IRB and Sponsor (see Section 14) approval before any changes are made in the 
research study, except when necessary to eliminate hazards to the patients/subjects. Comply with all 
other requirements regarding the obligations of clinical investigators and all other pertinent 
requirements listed in § 21 CFR part 312. 
 
15.2 Study Principal Investigator 
The Study Principal Investigator is responsible for the conduct of the clinical trial, including overseeing 
that sponsor responsibilities as defined in § 21 CFR 312. Subpart D is executed in accordance with 
federal regulations.  
15.3 Protocol Management Team (PMT) 
The Protocol Management Team (PMT) minimally consisting of the study principal investigator, site 
principal investigators, collaborating investigators, the research nurse, the clinical research 
associate/coordinator, and the study biostatistician is responsible for ongoing monitoring of the data 
and safety of this study, including implementation of the stopping rules for safety and efficacy. 
The PMT will meet (in person or via teleconference) at least monthly to review study status. This review 
will include, but not be limited to, reportable AEs and UPs, and an update of the ongoing study summary 
that describes study progress in terms of the study schema. The meeting will be a forum to discuss study 
related issues including accrual, SAE/AEs experienced, study response, deviations/violations and study 
management issues. The appropriateness of further subject enrollment and the specific intervention for 
subsequent subject enrollment are addressed. 

 
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PMT reports will be prepared at COH using the PMT report form in IRIS. Since there is an Independent 
DMC for this study, PMT reports will be exempt from COH Committee review, but stored as ‘submitted’ 
in IRIS. PMT reports will be retrieved by the study CRA for review by the Independent DMC as necessary.  
 
15.4 Monitoring  
The Investigator/Institution will permit direct access of the study monitors and appropriate regulatory 
authorities to the study data and to the corresponding source data and documents to verify the 
accuracy of this data. 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will also 
ensure that the monitor or other compliance or quality assurance reviewer is given access to all the 
above noted study-related documents and study related facilities (e.g. pharmacy, diagnostic laboratory, 
etc.), and has adequate space to conduct the monitoring visit. 
Clinical site monitoring is conducted to ensure that the rights of human subjects are protected, that the 
study is implemented in accordance with the protocol and regulatory requirements, and that the quality 
and integrity of study data and data collection methods are maintained.  Monitoring for this study will 
be performed by the City of Hope Office of Clinical Trials Auditing and Monitoring (OCTAM),  according 
to the COH OCTAM SOP which  is provided as a supplement to this document. 
Staff from OCTAM will conduct monitoring activities and provide reports of the findings and associated 
action items in accordance with the City of Hope Office of Clinical Trials Auditing and Monitoring SOP.  
Documentation of monitoring activities and findings will be provided to the site study teams, the site PI, 
study PI and Independent DMC. 
 
15.5 Quality Assurance  
The City of Hope Clinical Research Information Support will provide quality assurance as detailed in the 
COH DCC Operations Plan Manual.  
 
 

 
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16.0 ETHICAL AND REGULATORY CONSIDERATIONS 
16.1 Ethical Standard 
This study will be conducted in conformance with the principles set forth in The Belmont Report: Ethical 
Principles and Guidelines for the Protection of Human Subjects of Research (US National Commission for 
the Protection of Human Subjects of Biomedical and Behavioral Research, April 18, 1979) and the 
Declaration of Helsinki. 
16.2 Regulatory Compliance 
This study is to be conducted in compliance with the IRB approved protocol and according to the 
following considerations: 
• US Code of Federal Regulations (CFR) governing clinical study conduct 
o Title 21 Part 11 – Electronic Records; Electronic Signatures  
o Title 21 Part 50 – Protection of Human Subjects  
o Title 21 Part 54 – Financial Disclosure by Clinical Investigators  
o Title 21 Part 56 – Institutional Review Boards  
o Title 21 Part 58 – Good Laboratory Practice for Nonclinical Laboratory Studies  
o Title 21 Part 312 – Investigational New Drug Application  
o Title 45 Part 46 – Protection of Human Subjects  
• US Federal legislation, including but not limited to 
o Health Insurance Portability and Accountability Act of 1996  
o Section 801 of the Food and Drug Administration Amendments Act   
• Applicable state and local laws. For research occurring in California, this includes but is not 
limited to State of California Health and Safety Code, Title 17 
• Applicable institutional research policies and procedures  
 
16.3 Institutional Review Board 
Each participating institution must provide for the review and approval of this protocol and the 
associated informed consent documents by an appropriate IRB holding a current US Federal wide 
Assurance issued by and registered with the Office for Human Research Protections (OHRP). Any 
documents that the IRB may need to fulfill its responsibilities (such as protocol, protocol amendments, 
Investigator’s Brochure, consent forms or other pertinent information) will be submitted to the IRB.  The 
IRB’s written unconditional approval of the study protocol and the informed consent document will be 
in the possession of the Investigator, and, for sites external to COH, the possession of the coordinating 
center, before the study is initiated. The Investigator will obtain assurance of IRB/IEC compliance with 
regulations. 
The IRB will be informed of serious unexpected or unanticipated adverse experiences occurring during 
the study and any new information that may adversely affect patient safety or conduct of the study. 
Any amendment to the protocol document and accompanying informed consent document/template, 
as developed and provided by the Study PI, will require review and approval by the IRB before the 
changes are implemented in the study.  The protocol and consent will be reviewed and approved by the 
COH IRB before submission to a participating site IRB. 
 

 
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16.4 Informed Consent 
For a multi-site study, each participating institution will be provided with a model informed consent 
form. Each institution may revise or add information to comply with local and/or institutional 
requirements, but may not remove procedural or risk content from the model consent form. 
Furthermore, prior to submission to the IRB (initial submission and amendments), the consent and 
accompanying HIPAA form, if separate to the consent, must be reviewed and approved by the Data 
Coordinating Center. 
After the study has been fully explained, written informed consent will be obtained from either the 
patient or his/her guardian or legal representative before study participation.  The method of obtaining 
and documenting the informed consent and the contents of the consent must comply with the ICH-GCP 
and all applicable regulatory requirements. 
Before implementing any study procedure, informed consent shall be documented by the use of a 
written consent form approved by the IRB/IEC and signed and dated by the patient or the patient’s 
legally authorized representative at the time of consent.  A copy of the signed informed consent will be 
given to the patient or patient’s legally authorized representative.  The original signed consent must be 
maintained by the Site Investigator and available for inspection sponsor designated representatives, or 
regulatory authority at any time. 
Informed consent is a process that is initiated prior to the individual agreeing to participate in the study 
and continues throughout study participation.  
 
16.5 Women, Minorities, Children, HIV-Positive Individuals (Special Populations) 
16.5.1 Inclusion of Women and Minorities 
The study is open anyone regardless of gender or ethnicity.  Efforts will be made to extend the accrual to 
a representative population, but in a trial which will accrue and randomize approximately 102 subjects, 
a balance must be struck between subject safety considerations and limitations on the number of 
individuals exposed to potentially toxic or ineffective treatments on the one hand and the need to 
explore gender, racial, and ethnic aspects of clinical research on the other.  If differences in outcome 
that correlate to gender, racial, or ethnic identity are noted, accrual may be expanded or additional 
studies may be performed to investigate those differences more fully. 
Women who are pregnant or plan to become pregnant are excluded from participation because CMV-
MVA Triplex has unknown properties and has not been explored in a developmental study in children.  
Because there is an unknown but potential risk for adverse events in nursing infants secondary to 
treatment of the mother with the administered vaccine, breastfeeding should be discontinued if the 
mother is enrolled on this study.   
16.5.2 Exclusion of Pediatric Patients 
Pediatric recipients (children <18 years old of age) are excluded from this study because insufficient data 
are available in adults to judge potential risks in children.  Additionally, vaccine dosage and the blood 
volume established for immune-monitoring in adults cannot be applicable for both adults and children. 
Finally, the risk of CMV complications is inversely related to age, and the inclusion of younger children 
could bias the endpoint observations.  

 
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16.5.3 Exclusion of HIV Positive Individuals 
Individuals who are positive for HIV are expected to have very different underlying immune functions 
and therefore may respond to the CMV-MVA Triplex vaccine differently from individuals negative for 
HIV.  
 
16.6 Participant Confidentiality 
Participant confidentiality is strictly held in trust by the investigators, study staff, and the sponsor(s) and 
their agents.  This confidentiality is extended to cover testing of biological samples in addition to any 
study information relating to participants. 
This research will be conducted in compliance with federal and state requirements relating to protected 
health information (PHI), including the requirements of the Health Insurance Portability and 
Accountability Act of 1996 (HIPAA).  HIPAA regulations require a signed subject authorization informing 
the subject of the nature of the PHI to be collected, who will have access to that information and why, 
who will use or disclose that information, ant the rights of a research participant to revoke their 
authorization for use of their PHI. In the event that a subject revokes authorization to collect or use PHI, 
the investigator, by regulation, retains the ability to use all information collected prior to the revocation 
of subject authorization.  For subjects that have revoked authorization to collect or use PHI, attempts 
should be made to obtain permission to collect at least vital status (i.e. that the subject is alive) at the 
end of their scheduled study period. 
Release of research results should preserve the privacy of medical information and must be carried out 
in accordance with Department of Health and Human Services Standards for Privacy of Individually 
Identifiable Health Information, 45 CFR 164.508. When results of this study are reported in medical 
journals or at meetings, identification of those taking part will not be disclosed and no identifiers will be 
used. 
Medical records of subjects will be securely maintained in the strictest confidence, according to current 
legal requirements.  Data will be entered, analyzed and stored in encrypted, password protected, secure 
computers that meet all HIPAA requirements.   All data capture records, drug accountability records, 
study reports and communications will identify the patient by initials and the assigned patient number. 
Source documents provided to coordinating center for the purpose of auditing or monitoring will be de-
identified and labeled with the study number, subject ID, and patient initials. 
The investigator/institution will permit direct access to source data and documents by sponsor 
representatives, the FDA, and other applicable regulatory authorities.  The access may consist of trial-
related monitoring, including remote monitoring, audits, IRB/IEC reviews, and FDA/regulatory authority 
inspections.  The patient’s confidentiality will be maintained and will not be made publicly available to 
the extent permitted by the applicable laws and regulations. 
 
16.7 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or financial 
gain greater than the minimum allowable by their institution, etc.) must have the conflict reviewed by a 
properly constituted Conflict of Interest Committee with a Committee-sanctioned conflict management 
plan that has been reviewed and approved by the study Sponsor prior to participation in this study.  All 
City of Hope investigators will follow the City of Hope conflict of interest policy. 

 
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A financial disclosure form will be obtained from each external DMC member.  The SMT will maintain a 
copy of the form with study DMC files.  The DMC members will be required to provide annual updates of 
when their financial interests change during the life of the clinical study.  DMC members who develop 
serious conflicts of interest that could impact objectivity may be removed from the DMC and will be 
replaced (see DMC Charter for further details).   
 
16.8 Financial Obligations, Compensation, and Reimbursement of Participants 
The investigational drug including CMV-MVA Triplex and the matched placebo will be provided free of 
charge by COH.   
Neither the research participant nor the insurance carrier will be responsible for the research 
procedures related to this study. 
The standard of care drugs or procedures provided during the course of study participation will be the 
responsibility of the research participant and/or the insurance carrier. The research participant will be 
responsible for all copayments, deductibles, and other costs of treatment and diagnostic procedures as 
set forth by the insurance carrier.  The research participant and/or the insurance carrier will be billed for 
the costs of treatment and diagnostic procedures in the same way as if the research participant were 
not in a research study.  In the event of physical injury to a research participant resulting from research 
procedures, appropriate medical treatment will be available at City of Hope to the injured research 
participant. City of Hope will not provide financial compensation in the event of physical injury to a 
research participant. The research participant will not receive reimbursement or payment for taking part 
in this study. 
 
16.9 Publication/Data Sharing  
Any part of the results of the study carried out under this protocol, nor any of the information provided 
by City of Hope for the purposes of performing the study, will be published or passed on to any third 
party without the written approval of Dr. Don J. Diamond.  Any investigator involved with this study is 
obligated to provide City of Hope with complete test results and all data derived from the study. 
The preparation and submission for publication of manuscripts containing the study results shall be in 
accordance with a process determined by mutual written agreement among the City of Hope and 
participating institutions.  The publication or presentation of any study results shall comply with all 
applicable privacy laws, including, but not limited to, the Health Insurance Portability and Accountability 
Act of 1996.  
This study will comply with the NIH Public Access Policy, which ensures that the public has access to the 
published results of NIH funded research. It requires scientists to submit final peer-reviewed journal 
manuscripts that arise from NIH funds to the digital archive PubMed Central upon acceptance for 
publication. 
In accordance with the U.S. Public Law 110-85 (Food and Drug Administration Amendments Act of 2007 
or FDAAA), Title VIII, Section 801, this trial will be registered onto ClinicalTrials.gov and results will be 
reported on ClinicalTrials.gov within 12 months of the estimated or actual completion date of the trial, 
whichever date is earlier.  
The results should be made public within 24 months of reaching the end of the study. The end of the 
study is the time point at which the last data items are to be reported, or after the outcome data are 

 
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sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate and Study 
Duration. If a report is planned to be published in a peer-reviewed journal, then that initial release may 
be an abstract that meets the requirements of the International Committee of Medical Journal Editors. A 
full report of the outcomes should be made public no later than three (3) years after the end of the 
study.  
 

 
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17.0 REFERENCES 
1. 
Chee, M.S., et al., Analysis of the protein-coding content of the sequence of human 
cytomegalovirus strain AD169. Curr Top Microbiol Immunol, 1990. 154: p. 125-69. 
2. 
Applebaum, F.R., et al., Nonbacterial nonfungal pneumonia following marrow transplantation in 
100 identical twins. Transplantation, 1982. 33(3): p. 265-8. 
3. 
Quinnan, G.V., Jr., et al., Cytotoxic T cells in cytomegalovirus infection: HLA-restricted T- 
lymphocyte and non-T-lymphocyte cytotoxic responses correlate with recovery from 
cytomegalovirus infection in bone-marrow- transplant recipients. N.Engl.J.Med., 1982. 307: p. 7-
13. 
4. 
Reusser, P., et al., Randomized multicenter trial of foscarnet versus ganciclovir for preemptive 
therapy of cytomegalovirus infection after allogeneic stem cell transplantation. Blood, 2002. 
99(4): p. 1159-1164. 
5. 
Winston, D.J., et al., Infectious complications of human bone marrow transplantation. Medicine 
(Baltimore), 1979. 58(1): p. 1-31. 
6. 
Boeckh, M. and W.G. Nichols, The impact of cytomegalovirus serostatus of donor and recipient 
before hematopoietic stem cell transplantation in the era of antiviral prophylaxis and preemptive 
therapy. Blood, 2004. 103(6): p. 2003-2008. 
7. 
Nichols, W.G., et al., High Risk of Death Due to Bacterial and Fungal Infection among 
Cytomegalovirus (CMV)-Seronegative Recipients of Stem Cell Transplants from Seropositive 
Donors: Evidence for Indirect Effects of Primary CMV Infection. J.Infect.Dis., 2002. 185(3): p. 273-
282. 
8. 
Hillyer, C.D., D.R. Snydman, and E.M. Berkman, The risk of cytomegalovirus infection in solid 
organ and bone marrow transplant recipients: transfusion of blood products. Transfusion, 1990. 
30(7): p. 659-66. 
9. 
Zaia, J.A., Epidemiology and Pathogenesis of Cytomegalovirus Disease. Semin Hematol, 1990. 27: 
p. 5-10. 
10. 
Gallant, J.E., et al., Incidence and natural history of cytomegalovirus disease in patients with 
advanced human immunodeficiency virus disease treated with zidovudine. The Zidovudine 
Epidemiology Study Group. J Infect Dis, 1992. 166(6): p. 1223-7. 
11. 
Khanna, R. and D.J. Diamond, Human cytomegalovirus vaccine: time to look for alternative 
options. Trends Mol.Med., 2006. 12(1): p. 26-33. 
12. 
Mayr, A., [Historical review of smallpox, the eradication of smallpox and the attenuated 
smallpox MVA vaccine]. Berl Munch Tierarztl Wochenschr, 1999. 112(9): p. 322-8. 
13. 
Walsh, S.R., et al., Safety and immunogenicity of modified vaccinia ankara in hematopoietic stem 
cell transplant recipients: a randomized, controlled trial. J Infect Dis, 2013. 207(12): p. 1888-97. 
14. 
La Rosa, C., et al., Preclinical development of an adjuvant-free peptide vaccine with activity 
against CMV pp65 in HLA transgenic mice. Blood, 2002. 100(10): p. 3681-9. 
15. 
La Rosa, C., et al., Clinical evaluation of safety and immunogenicity of PADRE-cytomegalovirus 
(CMV) and tetanus-CMV fusion peptide vaccines with or without PF03512676 adjuvant. J Infect 
Dis, 2012. 205(8): p. 1294-304. 
16. 
Nakamura R., L.R.C., Longmate J., O'Donnell M., Cai J., Farol L., Salhotra M., Snyder D.S.,  Drake 
J., Slape C., Merson J., Zaia J.A., Forman S.J., Diamond DJ, Randomized Trial of a Novel CMV 
Vaccine (CMVPepVax) after Allogeneic HCT: Elevated CMV-Specific Immune Response, Reduction 

 
Page 73 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
in Chronic GvHD and CMV Reactivation Only in Vaccine Arm Patients. Blood 2014. 124(21): p. 
183. 
17. 
Wang, Z., et al., Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T 
cells. Blood, 2004. 104(3): p. 847-56. 
18. 
Aubert, G., et al., Cytomegalovirus-specific cellular immune responses and viremia in recipients 
of allogeneic stem cell transplants. J.Infect.Dis., 2001. 184(8): p. 955-963. 
19. 
Lacey, S.F., et al., Characterization of cytotoxic function of CMV-pp65-specific CD8+ T- 
lymphocytes identified by HLA tetramers in recipients and donors of stem-cell transplants. 
Transplantation, 2002. 74(5): p. 722-732. 
20. 
Gratama, J.W., et al., Immune monitoring with iTAg MHC Tetramers for prediction of recurrent or 
persistent cytomegalovirus infection or disease in allogeneic hematopoietic stem cell transplant 
recipients: a prospective multicenter study. Blood, 2010. 116(10): p. 1655-1662. 
21. 
Villacres, M.C., et al., Human immunodeficiency virus-infected patients receiving highly active 
antiretroviral therapy maintain activated cd8+ t cell subsets as a strong adaptive immune 
response to cytomegalovirus. J.Infect.Dis., 2001. 184(3): p. 256-267. 
22. 
Paley, M.A., et al., Progenitor and terminal subsets of CD8+ T cells cooperate to contain chronic 
viral infection. Science, 2012. 338(6111): p. 1220-1225. 
23. 
Hertoghs, K.M., et al., Molecular profiling of cytomegalovirus-induced human CD8+ T cell 
differentiation. J.Clin.Invest, 2010. 120(11): p. 4077-4090. 
24. 
Foley, B., et al., Human cytomegalovirus (CMV)-induced memory-like NKG2C(+) NK cells are 
transplantable and expand in vivo in response to recipient CMV antigen. J.Immunol., 2012. 
189(10): p. 5082-5088. 
25. 
Foley, B., et al., Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting 
increase in educated NKG2C+ natural killer cells with potent function. Blood, 2012. 119(11): p. 
2665-2674. 
26. 
Lopez-Verges, S., et al., Expansion of a unique CD57(+)NKG2Chi natural killer cell subset during 
acute human cytomegalovirus infection. Proc.Natl.Acad.Sci.U.S.A, 2011. 108(36): p. 14725-
14732. 
27. 
Kuijpers, T.W., et al., Human NK cells can control CMV infection in the absence of T cells. Blood, 
2008. 112(3): p. 914-915. 
28. 
Zhou, W., et al., Impact of donor CMV status on viral infection and reconstitution of 
multifunction CMV-specific T cells in CMV-positive transplant recipients. Blood, 2009. 113(25): p. 
6465-76. 
29. 
Przepiorka, D., et al., 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow 
Transplant, 1995. 15(6): p. 825-8. 
30. 
Bensinger, W.I., et al., Transplantation of bone marrow as compared with peripheral-blood cells 
from HLA-identical relatives in patients with hematologic cancers. N.Engl.J.Med., 2001. 344(3): p. 
175-181. 
31. 
Sylwester, A.W., et al., Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells 
dominate the memory compartments of exposed subjects. J.Exp.Med., 2005. 202(5): p. 673-685. 
32. 
Steininger, C., Clinical relevance of cytomegalovirus infection in patients with disorders of the 
immune system. Clin.Microbiol.Infect., 2007. 13(10): p. 953-963. 
33. 
Limaye, A.P., et al., Impact of cytomegalovirus in organ transplant recipients in the era of 
antiviral prophylaxis. Transplantation, 2006. 81(12): p. 1645-1652. 

 
Page 74 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
34. 
Griffiths, P., et al., Contemporary management of cytomegalovirus infection in transplant 
recipients: guidelines from an IHMF workshop, 2007. Herpes., 2008. 15(1): p. 4-12. 
35. 
Boeckh, M., et al., Cytomegalovirus in hematopoietic stem cell transplant recipients: Current 
status, known challenges, and future strategies. Biol.Blood Marrow Transplant., 2003. 9(9): p. 
543-558. 
36. 
Tomblyn, M., et al., Guidelines for preventing infectious complications among hematopoietic cell 
transplantation recipients: a global perspective. Biol.Blood Marrow Transplant, 2009. 15(10): p. 
1143-1238. 
37. 
Zerr, D.M., et al., HHV-6 reactivation and its effect on delirium and cognitive functioning in 
hematopoietic cell transplantation recipients. Blood, 2011. 117(19): p. 5243-5249. 
38. 
Reusser, P., Oral valganciclovir: a new option for treatment of cytomegalovirus infection and 
disease in immunocompromised hosts. Expert.Opin.Investig.Drugs, 2001. 10(9): p. 1745-1753. 
39. 
Ljungman, P., M. Hakki, and M. Boeckh, Cytomegalovirus in hematopoietic stem cell transplant 
recipients. Infect.Dis.Clin.North Am., 2010. 24(2): p. 319-337. 
40. 
Hakki, M., et al., Immune reconstitution to cytomegalovirus after allogeneic hematopoietic stem 
cell transplantation: impact of host factors, drug therapy, and subclinical reactivation. Blood, 
2003. 102(8): p. 3060-3067. 
41. 
Nguyen, Q., et al., Late cytomegalovirus pneumonia in adult allogeneic blood and marrow 
transplant recipients. Clin.Infect.Dis., 1999. 28(3): p. 618-623. 
42. 
Razonable, R.R., et al., Allograft rejection predicts the occurrence of late-onset cytomegalovirus 
(CMV) disease among CMV-mismatched solid organ transplant patients receiving prophylaxis 
with oral ganciclovir. J.Infect.Dis., 2001. 184(11): p. 1461-1464. 
43. 
Boeckh, M., et al., Late cytomegalovirus disease and mortality in recipients of allogeneic 
hematopoietic stem cell transplants: importance of viral load and T-cell immunity. Blood, 2003. 
101(2): p. 407-414. 
44. 
Peggs, K.S. and S. Mackinnon, Immune reconstitution following haematopoietic stem cell 
transplantation. Br.J.Haematol., 2004. 124(4): p. 407-420. 
45. 
Singh, N., Delayed occurrence of cytomegalovirus disease in organ transplant recipients receiving 
antiviral 
prophylaxis: 
are 
we 
winning 
the 
battle 
only 
to 
lose 
the 
war? 
Eur.J.Clin.Microbiol.Infect.Dis., 2002. 21(12): p. 902. 
46. 
McConkey, S.J., et al., Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by 
recombinant modified vaccinia virus Ankara in humans. Nat Med, 2003. 9(6): p. 729-35. 
47. 
Hawkridge, T., et al., Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in 
healthy adults in South Africa. J Infect Dis, 2008. 198(4): p. 544-52. 
48. 
Stittelaar, K.J., et al., Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed 
macaques. Vaccine, 2001. 19(27): p. 3700-9. 
49. 
Cosma, A., et al., Therapeutic vaccination with MVA-HIV-1 nef elicits Nef-specific T-helper cell 
responses in chronically HIV-1 infected individuals. Vaccine, 2003. 22(1): p. 21-9. 
50. 
Wyatt, L.S., et al., Highly attenuated smallpox vaccine protects mice with and without immune 
deficiencies against pathogenic vaccinia virus challenge. Proc Natl Acad Sci U S A, 2004. 101(13): 
p. 4590-5. 
51. 
Moss, B., et al., Host range restricted, non-replicating vaccinia virus vectors as vaccine 
candidates. Adv Exp Med Biol, 1996. 397: p. 7-13. 

 
Page 75 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
52. 
Khan, N., et al., T cell recognition patterns of immunodominant cytomegalovirus antigens in 
primary and persistent infection. J Immunol, 2007. 178(7): p. 4455-65. 
53. 
van de Berg, P.J., et al., Cytomegalovirus infection reduces telomere length of the circulating T 
cell pool. J Immunol, 2010. 184(7): p. 3417-23. 
54. 
Gandhi, M.K., et al., Late diversification in the clonal composition of human cytomegalovirus-
specific CD8+ T cells following allogeneic hemopoietic stem cell transplantation. Blood, 2003. 
102(9): p. 3427-38. 
55. 
Khan, N., et al., Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the 
response to a coresident EBV infection. J Immunol, 2004. 173(12): p. 7481-9. 
56. 
Elkington, R., et al., Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals 
broad and multispecific reactivities in healthy virus carriers. J Virol, 2003. 77(9): p. 5226-40. 
57. 
Boppana, S.B. and W.J. Britt, Recognition of human cytomegalovirus gene products by HCMV-
specific cytotoxic T cells. Virology, 1996. 222(1): p. 293-6. 
58. 
McLaughlin-Taylor, E., et al., Identification of the major late human cytomegalovirus matrix 
protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. J Med Virol, 
1994. 43(1): p. 103-10. 
59. 
Wills, M.R., et al., The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is 
dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-
specific CTL. J Virol, 1996. 70(11): p. 7569-79. 
60. 
Lacey, S.F., et al., Functional comparison of T cells recognizing cytomegalovirus pp65 and 
intermediate-early antigen polypeptides in hematopoietic stem-cell transplant and solid organ 
transplant recipients. J Infect Dis, 2006. 194(10): p. 1410-21. 
61. 
Gyulai, Z., et al., Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-
Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific 
CTLs. J Infect Dis, 2000. 181(5): p. 1537-46. 
62. 
Kern, F., et al., Target structures of the CD8(+)-T-cell response to human cytomegalovirus: the 
72-kilodalton major immediate-early protein revisited. J Virol, 1999. 73(10): p. 8179-84. 
63. 
Khan, N., et al., Comparative analysis of CD8+ T cell responses against human cytomegalovirus 
proteins pp65 and immediate early 1 shows similarities in precursor frequency, oligoclonality, 
and phenotype. J Infect Dis, 2002. 185(8): p. 1025-34. 
64. 
Wang, Z., et al., A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular 
immunity in an MVA vaccine vector. Virology, 2008. 377(2): p. 379-90. 
65. 
Bunde, T., et al., Protection from cytomegalovirus after transplantation is correlated with 
immediate early 1-specific CD8 T cells. J Exp Med, 2005. 201(7): p. 1031-6. 
66. 
Nickel, P., et al., High levels of CMV-IE-1-specific memory T cells are associated with less 
alloimmunity and improved renal allograft function. Transpl Immunol, 2009. 20(4): p. 238-42. 
67. 
Berencsi, K., et al., A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 
induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects. J Infect Dis, 
2001. 183(8): p. 1171-9. 
68. 
Bernstein, D.I., et al., Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine 
for cytomegalovirus in CMV seronegative adult volunteers. Vaccine, 2009. 28(2): p. 484-93. 
69. 
Selinsky, C., et al., A DNA-based vaccine for the prevention of human cytomegalovirus-associated 
diseases. Hum Vaccin, 2005. 1(1): p. 16-23. 

 
Page 76 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
70. 
Gratama, J.W., et al., Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T 
lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at 
risk for progressive CMV infection. Blood, 2001. 98(5): p. 1358-64. 
71. 
Cwynarski, K., et al., Direct visualization of cytomegalovirus-specific T-cell reconstitution after 
allogeneic stem cell transplantation. Blood, 2001. 97(5): p. 1232-40. 
72. 
Lacey, S.F., et al., Characterization of cytotoxic function of CMV-pp65-specific CD8+ T-
lymphocytes identified by HLA tetramers in recipients and donors of stem-cell transplants. 
Transplantation, 2002. 74(5): p. 722-32. 
73. 
Gallez-Hawkins, G., et al., Cytomegalovirus immune reconstitution occurs in recipients of 
allogeneic hematopoietic cell transplants irrespective of detectable cytomegalovirus infection. 
Biol Blood Marrow Transplant, 2005. 11(11): p. 890-902. 
74. 
Pourgheysari, B., et al., Early reconstitution of effector memory CD4+ CMV-specific T cells 
protects against CMV reactivation following allogeneic SCT. Bone Marrow Transplant, 2009. 
43(11): p. 853-61. 
75. 
Lilleri, D., et al., Human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in adult 
allogeneic hematopoietic stem cell transplant recipients and immune control of viral infection. 
Haematologica, 2008. 93(2): p. 248-56. 
76. 
Luo, X.H., et al., Protective immunity transferred by infusion of cytomegalovirus-specific CD8(+) T 
cells within donor grafts: its associations with cytomegalovirus reactivation following 
unmanipulated allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow 
Transplant, 2010. 16(7): p. 994-1004. 
77. 
Gratama, J.W., et al., Immune monitoring with iTAg MHC Tetramers for prediction of recurrent or 
persistent cytomegalovirus infection or disease in allogeneic hematopoietic stem cell transplant 
recipients: a prospective multicenter study. Blood, 2010. 116(10): p. 1655-62. 
78. 
Plotkin, S.A., et al., Effect of Towne live virus vaccine on cytomegalovirus disease after renal 
transplant. A controlled trial. Ann Intern Med, 1991. 114(7): p. 525-31. 
79. 
Scheinberg, P., et al., The transfer of adaptive immunity to CMV during hematopoietic stem cell 
transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the 
donor. Blood, 2009. 114(24): p. 5071-80. 
80. 
Ljungman, P., et al., Donor CMV serologic status and outcome of CMV-seropositive recipients 
after unrelated donor stem cell transplantation: an EBMT megafile analysis. Blood, 2003. 
102(13): p. 4255-60. 
81. 
Uzunel, M., et al., Unrelated versus related allogeneic stem cell transplantation after reduced 
intensity conditioning. Transplantation, 2006. 82(7): p. 913-9. 
82. 
Jamieson, B.D. and R. Ahmed, T cell memory. Long-term persistence of virus-specific cytotoxic T 
cells. J Exp Med, 1989. 169(6): p. 1993-2005. 
83. 
Matloubian, M., R.J. Concepcion, and R. Ahmed, CD4+ T cells are required to sustain CD8+ 
cytotoxic T-cell responses during chronic viral infection. J Virol, 1994. 68(12): p. 8056-63. 
84. 
Plotkin, S.A., et al., Protective effects of Towne cytomegalovirus vaccine against low-passage 
cytomegalovirus administered as a challenge. J Infect Dis, 1989. 159(5): p. 860-5. 
85. 
Boppana, S.B., et al., Intrauterine transmission of cytomegalovirus to infants of women with 
preconceptional immunity. N Engl J Med, 2001. 344(18): p. 1366-71. 
86. 
Gonczol, E. and S. Plotkin, Progress in vaccine development for prevention of human 
cytomegalovirus infection. Curr Top Microbiol Immunol, 1990. 154: p. 255-74. 

 
Page 77 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
87. 
Adler, S.P., et al., A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes 
for antibody responses to a live attenuated CMV vaccine (Towne). J Infect Dis, 1999. 180(3): p. 
843-6. 
88. 
Bernstein, D.I., et al., Effect of previous or simultaneous immunization with canarypox expressing 
cytomegalovirus (CMV) glycoprotein B (gB) on response to subunit gB vaccine plus MF59 in 
healthy CMV-seronegative adults. J Infect Dis, 2002. 185(5): p. 686-90. 
89. 
Pass, R.F., et al., A subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein 
B and a new adjuvant. J Infect Dis, 1999. 180(4): p. 970-5. 
90. 
Endresz, V., et al., Optimization of DNA immunization against human cytomegalovirus. Vaccine, 
2001. 19(28-29): p. 3972-80. 
91. 
Endresz, V., et al., Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific 
neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses 
by naked DNA immunization. Vaccine, 1999. 17(1): p. 50-8. 
92. 
Pande, H., et al., Direct DNA immunization of mice with plasmid DNA encoding the tegument 
protein pp65 (ppUL83) of human cytomegalovirus induces high levels of circulating antibody to 
the encoded protein. Scand J Infect Dis Suppl, 1995. 99: p. 117-20. 
93. 
Wloch, M.K., et al., Safety and immunogenicity of a bivalent cytomegalovirus DNA vaccine in 
healthy adult subjects. J Infect Dis, 2008. 197(12): p. 1634-42. 
94. 
Kharfan-Dabaja, M.A., et al., A novel therapeutic cytomegalovirus DNA vaccine in allogeneic 
haemopoietic stem-cell transplantation: a randomised, double-blind, placebo-controlled, phase 2 
trial. Lancet Infect Dis, 2012. 12(4): p. 290-9. 
95. 
Stickl, H., et al., [MVA vaccination against smallpox: clinical tests with an attenuated live vaccinia 
virus strain (MVA) (author's transl)]. Dtsch Med Wochenschr, 1974. 99(47): p. 2386-92. 
96. 
Mayr, A., et al., [The smallpox vaccination strain MVA: marker, genetic structure, experience 
gained with the parenteral vaccination and behavior in organisms with a debilitated defence 
mechanism (author's transl)]. Zentralbl Bakteriol [B], 1978. 167(5-6): p. 375-90. 
97. 
Redfield, R.R., et al., Disseminated vaccinia in a military recruit with human immunodeficiency 
virus (HIV) disease. N Engl J Med, 1987. 316(11): p. 673-6. 
98. 
Collier, L.H., Safety of recombinant vaccinia vaccines. Lancet, 1991. 337(8748): p. 1035-6. 
99. 
Meyer, H., G. Sutter, and A. Mayr, Mapping of deletions in the genome of the highly attenuated 
vaccinia virus MVA and their influence on virulence. J Gen Virol, 1991. 72 ( Pt 5): p. 1031-8. 
100. 
Antoine, G., et al., The complete genomic sequence of the modified vaccinia Ankara strain: 
comparison with other orthopoxviruses. Virology, 1998. 244(2): p. 365-96. 
101. 
Carroll, M.W. and B. Moss, Host range and cytopathogenicity of the highly attenuated MVA 
strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman 
mammalian cell line. Virology, 1997. 238(2): p. 198-211. 
102. 
Carroll, M.W., et al., Highly attenuated modified vaccinia virus Ankara (MVA) as an effective 
recombinant vector: a murine tumor model. Vaccine, 1997. 15(4): p. 387-94. 
103. 
Blanchard, T.J., et al., Modified vaccinia virus Ankara undergoes limited replication in human 
cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. J 
Gen Virol, 1998. 79 ( Pt 5): p. 1159-67. 
104. 
Sutter, G. and B. Moss, Nonreplicating vaccinia vector efficiently expresses recombinant genes. 
Proc Natl Acad Sci U S A, 1992. 89(22): p. 10847-51. 

 
Page 78 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
105. 
Ramirez, J.C., et al., Attenuated modified vaccinia virus Ankara can be used as an immunizing 
agent under conditions of preexisting immunity to the vector. J Virol, 2000. 74(16): p. 7651-5. 
106. 
Ramirez, J.C., M.M. Gherardi, and M. Esteban, Biology of attenuated modified vaccinia virus 
Ankara recombinant vector in mice: virus fate and activation of B- and T-cell immune responses 
in comparison with the Western Reserve strain and advantages as a vaccine. J Virol, 2000. 74(2): 
p. 923-33. 
107. 
Cottingham, M.G. and M.W. Carroll, Recombinant MVA vaccines: dispelling the myths. Vaccine, 
2013. 31(39): p. 4247-51. 
108. 
Harrop, R., et al., Cross-trial analysis of immunologic and clinical data resulting from phase I and 
II trials of MVA-5T4 (TroVax) in colorectal, renal, and prostate cancer patients. J Immunother, 
2010. 33(9): p. 999-1005. 
109. 
Goepfert, P.A., et al., Phase 1 safety and immunogenicity testing of DNA and recombinant 
modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis, 2011. 203(5): 
p. 610-9. 
110. 
Earl, P.L., et al., Immunogenicity of a highly attenuated MVA smallpox vaccine and protection 
against monkeypox. Nature, 2004. 428(6979): p. 182-5. 
111. 
de Waal, L., et al., Vaccination of infant macaques with a recombinant modified vaccinia virus 
Ankara expressing the respiratory syncytial virus F and G genes does not predispose for 
immunopathology. Vaccine, 2004. 22(8): p. 923-6. 
112. 
Moorthy, V.S., et al., Safety of DNA and modified vaccinia virus Ankara vaccines against liver-
stage P. falciparum malaria in non-immune volunteers. Vaccine, 2003. 21(17-18): p. 1995-2002. 
113. 
Prieur, E., et al., A Plasmodium falciparum candidate vaccine based on a six-antigen polyprotein 
encoded by recombinant poxviruses. Proc Natl Acad Sci U S A, 2004. 101(1): p. 290-5. 
114. 
Rochlitz, C., et al., Phase I immunotherapy with a modified vaccinia virus (MVA) expressing 
human MUC1 as antigen-specific immunotherapy in patients with MUC1-positive advanced 
cancer. J Gene Med, 2003. 5(8): p. 690-9. 
115. 
Song, G.Y., et al., An MVA vaccine overcomes tolerance to human p53 in mice and humans. 
Cancer Immunology, Immunotherapy, 2007. 56(8): p. 1193-205. 
116. 
Hardwick, N.R., et al., p53MVA therapy in patients with refractory gastrointestinal malignancies 
elevates p53-specific CD8+ T-cell responses. Clin Cancer Res, 2014. 20(17): p. 4459-70. 
117. 
Bender, B.S., et al., Oral immunization with a replication-deficient recombinant vaccinia virus 
protects mice against influenza. J Virol, 1996. 70(9): p. 6418-24. 
118. 
Sutter, G., et al., A recombinant vector derived from the host range-restricted and highly 
attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza 
virus. Vaccine, 1994. 12(11): p. 1032-40. 
119. 
Wyatt, L.S., et al., Development of a replication-deficient recombinant vaccinia virus vaccine 
effective against parainfluenza virus 3 infection in an animal model. Vaccine, 1996. 14(15): p. 
1451-8. 
120. 
Wyatt, L.S., et al., Priming and boosting immunity to respiratory syncytial virus by recombinant 
replication-defective vaccinia virus MVA. Vaccine, 1999. 18(5-6): p. 392-7. 
121. 
Men, R., et al., Immunization of rhesus monkeys with a recombinant of modified vaccinia virus 
Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance 
to dengue type 2 virus challenge. Vaccine, 2000. 18(27): p. 3113-22. 

 
Page 79 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
122. 
Nam, J.H., et al., Protection against lethal Japanese encephalitis virus infection of mice by 
immunization with the highly attenuated MVA strain of vaccinia virus expressing JEV prM and E 
genes. Vaccine, 1999. 17(3): p. 261-8. 
123. 
Degano, P., et al., Gene gun intradermal DNA immunization followed by boosting with modified 
vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the 
influenza and malaria models. Vaccine, 1999. 18(7-8): p. 623-32. 
124. 
Seth, A., et al., Immunization with a modified vaccinia virus expressing simian immunodeficiency 
virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is 
associated with reduction of viremia after SIV challenge. J Virol, 2000. 74(6): p. 2502-9. 
125. 
Allen, T.M., et al., Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral 
blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus 
Ankara boost regimen. J Immunol, 2000. 164(9): p. 4968-78. 
126. 
Hanke, T. and A. McMichael, Pre-clinical development of a multi-CTL epitope-based DNA prime 
MVA boost vaccine for AIDS. Immunol Lett, 1999. 66(1-3): p. 177-81. 
127. 
Hanke, T. and A.J. McMichael, Design and construction of an experimental HIV-1 vaccine for a 
year-2000 clinical trial in Kenya. Nat Med, 2000. 6(9): p. 951-5. 
128. 
Amara, R.R., et al., Control of a mucosal challenge and prevention of AIDS by a multiprotein 
DNA/MVA vaccine. Science, 2001. 292(5514): p. 69-74. 
129. 
Wussow, F., et al., A vaccine based on the rhesus cytomegalovirus UL128 complex induces 
broadly neutralizing antibodies in rhesus macaques. Journal of Virology, 2013. 87(3): p. 1322-32. 
130. 
Abel, K., et al., Vaccine-induced control of viral shedding following rhesus cytomegalovirus 
challenge in rhesus macaques. Journal of Virology, 2011. 85(6): p. 2878-90. 
131. 
Longmate, J., et al., Population coverage by HLA class-I restricted cytotoxic T-lymphocyte 
epitopes. Immunogenetics, 2001. 52(3-4): p. 165-73. 
132. 
Alp, N.J., et al., Fine specificity of cellular immune responses in humans to human 
cytomegalovirus immediate-early 1 protein. J Virol, 1991. 65(9): p. 4812-20. 
133. 
Gilbert, M.J., et al., Selective interference with class I major histocompatibility complex 
presentation of the major immediate-early protein following infection with human 
cytomegalovirus. J Virol, 1993. 67(6): p. 3461-9. 
134. 
La Rosa, C., et al., Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived 
from positional scanning synthetic combinatorial libraries. Blood, 2001. 97(6): p. 1776-86. 
135. 
Jin, X., et al., High frequency of cytomegalovirus-specific cytotoxic T-effector cells in HLA-A*0201-
positive subjects during multiple viral coinfections. J Infect Dis, 2000. 181(1): p. 165-75. 
136. 
Komanduri, K.V., et al., Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-
infected subjects. Virology, 2001. 279(2): p. 459-70. 
137. 
Gillespie, G.M., et al., Functional heterogeneity and high frequencies of cytomegalovirus-specific 
CD8(+) T lymphocytes in healthy seropositive donors. J Virol, 2000. 74(17): p. 8140-50. 
138. 
Retiere, C., et al., Generation of cytomegalovirus-specific human T-lymphocyte clones by using 
autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study 
the fine specificity of the antiviral response. J Virol, 2000. 74(9): p. 3948-52. 
139. 
Jacobson, M.A., et al., Results of a cytomegalovirus (CMV)-specific CD8+/interferon- gamma+ 
cytokine flow cytometry assay correlate with clinical evidence of protective immunity in patients 
with AIDS with CMV retinitis. J Infect Dis, 2004. 189(8): p. 1362-73. 

 
Page 80 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
140. 
La Rosa, C., et al., Longitudinal assessment of cytomegalovirus (CMV)-specific immune responses 
in liver transplant recipients at high risk for late CMV disease. J Infect Dis, 2007. 195(5): p. 633-
44. 
141. 
Spaete, R.R., R.C. Gehrz, and M.P. Landini, Human cytomegalovirus structural proteins. J Gen 
Virol, 1994. 75 ( Pt 12): p. 3287-308. 
142. 
Britt, W.J., et al., Cell surface expression of human cytomegalovirus (HCMV) gp55-116 (gB): use 
of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody 
response. J Virol, 1990. 64(3): p. 1079-85. 
143. 
Banks, T., et al., A major neutralizing domain maps within the carboxyl-terminal half of the 
cleaved cytomegalovirus B glycoprotein. J Gen Virol, 1989. 70 ( Pt 4): p. 979-85. 
144. 
Marshall, G.S., et al., An adenovirus recombinant that expresses the human cytomegalovirus 
major envelope glycoprotein and induces neutralizing antibodies. J Infect Dis, 1990. 162(5): p. 
1177-81. 
145. 
Macagno, A., et al., Isolation of human monoclonal antibodies that potently neutralize human 
cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex. 
Journal of Virology, 2010. 84(2): p. 1005-13. 
146. 
Kim, S., et al., Human cytomegalovirus IE1 protein activates AP-1 through a cellular protein 
kinase(s). J Gen Virol, 1999. 80 ( Pt 4): p. 961-9. 
147. 
Johnson, R.A., et al., Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 
protein-protein interaction and the possible functional consequences. J Gen Virol, 1999. 80 ( Pt 
5): p. 1293-303. 
148. 
Pajovic, S., et al., Identification of a viral kinase that phosphorylates specific E2Fs and pocket 
proteins. Mol Cell Biol, 1997. 17(11): p. 6459-64. 
149. 
Gallez-Hawkins, G., et al., Use of transgenic HLA A*0201/Kb and HHD II mice to evaluate 
frequency of cytomegalovirus IE1-derived peptide usage in eliciting human CD8 cytokine 
response. J Virol, 2003. 77(7): p. 4457-62. 
150. 
Drexler, I., et al., Highly attenuated modified vaccinia virus Ankara replicates in baby hamster 
kidney cells, a potential host for virus propagation, but not in various human transformed and 
primary cells. J Gen Virol, 1998. 79 ( Pt 2): p. 347-52. 
151. 
Scheiflinger, F., F.G. Falkner, and F. Dorner, Evaluation of the thymidine kinase (tk) locus as an 
insertion site in the highly attenuated vaccinia MVA strain. Arch Virol, 1996. 141(3-4): p. 663-9. 
152. 
Chakrabarti, S., J.R. Sisler, and B. Moss, Compact, synthetic, vaccinia virus early/late promoter 
for protein expression. Biotechniques, 1997. 23(6): p. 1094-7. 
153. 
Carroll, M.W. and B. Moss, E. coli beta-glucuronidase (GUS) as a marker for recombinant 
vaccinia viruses. Biotechniques, 1995. 19(3): p. 352-4, 356. 
154. 
Moss, B., Expression of proteins in mammalian cells using vaccina virus vectors.  . Current 
Protocols in Immunology 1998, New York Greene Publishing. 16.15.1-16.21.9. 
155. 
Riddell, S.R., et al., T-cell mediated rejection of gene-modified HIV-specific cytotoxic T 
lymphocytes in HIV-infected patients. Nat Med, 1996. 2(2): p. 216-23. 
156. 
Berger, C., et al., Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of 
gene-modified autologous T cells in a nonhuman primate model. J Virol, 2001. 75(2): p. 799-808. 
157. 
Scheiflinger, F., F. Dorner, and F.G. Falkner, Transient marker stabilisation: a general procedure 
to construct marker-free recombinant vaccinia virus. Arch Virol, 1998. 143(3): p. 467-74. 

 
Page 81 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
158. 
Wang, Z., et al., Vaccine properties of a novel marker gene-free recombinant modified vaccinia 
Ankara expressing immunodominant CMV antigens pp65 and IE1. Vaccine, 2007. 25(6): p. 1132-
41. 
159. 
Krishnan, A., et al., A novel approach to evaluate the immunogenicity of viral antigens of clinical 
importance in HLA transgenic murine models. Immunology Letters, 2008. 120(1-2): p. 108-16. 
160. 
Wang, Z., et al., Modified H5 promoter improves stability of insert genes while maintaining 
immunogenicity during extended passage of genetically engineered MVA vaccines. Vaccine, 
2010. 28(6): p. 1547-57. 
161. 
Krieg, A.M., CpG motifs in bacterial DNA and their immune effects. Annu.Rev.Immunol., 2002. 
20: p. 709-760. 
162. 
Davila, E., et al., Creating space: an antigen-independent, CpG-induced peripheral expansion of 
naive and memory T lymphocytes in a full T-cell compartment. Blood, 2002. 100(7): p. 2537-
2545. 
163. 
Kuo, C.C., S.M. Liang, and C.M. Liang, CpG-B oligodeoxynucleotide promotes cell survival via up-
regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation. 
J.Biol.Chem., 2006. 281(50): p. 38200-38207. 
164. 
Woolfrey, A., et al., HLA-C antigen mismatch is associated with worse outcome in unrelated 
donor peripheral blood stem cell transplantation. Biol Blood Marrow Transplant, 2011. 17(6): p. 
885-92. 
165. 
Saber, W., et al., Outcomes after matched unrelated donor versus identical sibling hematopoietic 
cell transplantation in adults with acute myelogenous leukemia. Blood, 2012. 119(17): p. 3908-
16. 
166. 
Bacigalupo, A., Matched and mismatched unrelated donor transplantation: is the outcome the 
same as for matched sibling donor transplantation? Hematology Am Soc Hematol Educ Program, 
2012. 2012: p. 223-9. 
167. 
Anasetti, C., What are the most important donor and recipient factors affecting the outcome of 
related and unrelated allogeneic transplantation? Best Pract Res Clin Haematol, 2008. 21(4): p. 
691-7. 
168. 
Sorror, M.L., et al., Long-term outcomes among older patients following nonmyeloablative 
conditioning and allogeneic hematopoietic cell transplantation for advanced hematologic 
malignancies. JAMA, 2011. 306(17): p. 1874-1883. 
169. 
Warlick, E., et al., Reduced intensity conditioning is superior to nonmyeloablative conditioning for 
older chronic myelogenous leukemia patients undergoing hematopoietic cell transplant during 
the tyrosine kinase inhibitor era. Blood, 2012. 119(17): p. 4083-4090. 
170. 
Wieand, S., G. Schroeder, and J.R. O'Fallon, Stopping when the experimental regimen does not 
appear to help. Stat.Med., 1994. 13(13-14): p. 1453-1458. 
171. 
Paczesny, S., et al., A biomarker panel for acute graft-versus-host disease. Blood, 2009. 113(2): p. 
273-8. 
172. 
Levine, J.E., et al., Acute graft-versus-host disease biomarkers measured during therapy can 
predict treatment outcomes: a Blood and Marrow Transplant Clinical Trials Network study. 
Blood, 2012. 119(16): p. 3854-60. 
173. 
Paczesny, S., et al., Elafin is a biomarker of graft-versus-host disease of the skin. Sci Transl Med, 
2010. 2(13): p. 13ra2. 
174. 
Ferrara, J.L., et al., Regenerating islet-derived 3-alpha is a biomarker of gastrointestinal graft-
versus-host disease. Blood, 2011. 118(25): p. 6702-8. 

 
Page 82 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
175. 
Vander Lugt, M.T., et al., ST2 as a marker for risk of therapy-resistant graft-versus-host disease 
and death. N Engl J Med, 2013. 369(6): p. 529-39. 
176. 
Kennedy, G.A., et al., Addition of interleukin-6 inhibition with tocilizumab to standard graft-
versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial. 
Lancet Oncol, 2014. 15(13): p. 1451-9. 
177. 
Sarantopoulos, S., et al., Altered B-cell homeostasis and excess BAFF in human chronic graft-
versus-host disease. Blood, 2009. 113(16): p. 3865-74. 
178. 
Lacey, S.F., D.J. Diamond, and J.A. Zaia, Assessment of cellular immunity to human 
cytomegalovirus in recipients of allogeneic stem cell transplants. Biol Blood Marrow Transplant, 
2004. 10(7): p. 433-47. 
179. 
Lacey, S.F., et al., Simultaneous reconstitution of multiple cytomegalovirus-specific CD8+ cell 
populations with divergent functionality in hematopoietic stem-cell transplant recipients. J Infect 
Dis, 2005. 191(6): p. 977-84. 
180. 
Upfal, M.J. and S. Cinti, Smallpox vaccination and adverse cardiac events. Emerging Infectious 
Diseases, 2004. 10(5): p. 961-2; discussion 962. 
181. 
Elizaga, M.L., et al., Prospective surveillance for cardiac adverse events in healthy adults 
receiving modified vaccinia Ankara vaccines: a systematic review. PLoS One, 2013. 8(1): p. 
e54407. 
182. 
Krishnan, A., et al., Programmed death-1 receptor and interleukin-10 in liver transplant 
recipients at high risk for late cytomegalovirus disease. Transpl Infect Dis, 2010. 12(4): p. 363-70. 
183. 
Wolfl, M., et al., Activation-induced expression of CD137 permits detection, isolation, and 
expansion of the full repertoire of CD8+ T cells responding to antigen without requiring 
knowledge of epitope specificities. Blood, 2007. 110(1): p. 201-10. 
184. 
Nakamura, R., et al., Ex vivo detection of CD8 T cells specific for H-Y minor histocompatibility 
antigens in allogeneic hematopoietic stem cell transplant recipients. Transpl Immunol, 2014. 
30(4): p. 128-35. 
185. 
La Rosa, C., et al., Primary response against cytomegalovirus during antiviral prophylaxis with 
valganciclovir, in solid organ transplant recipients. Transpl Int, 2011. 24(9): p. 920-31. 
186. 
Israyelyan, A., et al., Detection and preliminary characterization of CD8+T lymphocytes specific 
for Wilms' tumor antigen in patients with non-Hodgkin lymphoma. Leuk Lymphoma, 2013. 
54(11): p. 2490-9. 
187. 
Taub, D.D., et al., Immunity from smallpox vaccine persists for decades: a longitudinal study. Am 
J Med, 2008. 121(12): p. 1058-64. 
188. 
Kennedy, R.B., et al., The immunology of smallpox vaccines. Curr Opin Immunol, 2009. 21(3): p. 
314-20. 
189. 
Cosma, A., et al., Neutralization assay using a modified vaccinia virus Ankara vector expressing 
the green fluorescent protein is a high-throughput method to monitor the humoral immune 
response against vaccinia virus. Clin Diagn Lab Immunol, 2004. 11(2): p. 406-10. 
190. 
Wu, Y., et al., Reactive impurities in excipients: profiling, identification and mitigation of drug-
excipient incompatibility. AAPS PharmSciTech, 2011. 12(4): p. 1248-63. 
191. 
La Rosa, C., et al., Programmed death-1 expression in liver transplant recipients as a prognostic 
indicator of cytomegalovirus disease. J.Infect.Dis., 2008. 197(1): p. 25-33. 

 
Page 83 of 91 
IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
192. 
Elmaagacli, A.H., et al., Early human cytomegalovirus replication after transplantation is 
associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in 
acute myeloid leukemia patients. Blood, 2011. 118(5): p. 1402-1412. 
193. 
Green, M.L., et al., CMV reactivation after allogeneic HCT and relapse risk: evidence for early 
protection in acute myeloid leukemia. Blood, 2013. 122(7): p. 1316-1324. 
194. 
Rubinstein, L.V., et al., Design issues of randomized phase II trials and a proposal for phase II 
screening trials. J.Clin.Oncol., 2005. 23(28): p. 7199-7206. 
 
 
 
 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
APPENDIX A: ACUTE GVHD STAGING 
1994 Keystone Consensus Criteria 
Organ Staging of Clinical Acute GVHD 
Skin 
Lower GI 
Upper GI 
Liver (Total Bilb) 
0- No Rash 
 I- Maculopapular rash, 
<25% of body surface 
 II- Maculopapular rash, 
25-50% of body surface 
 III- Rash on >50% of body 
surface, or generalized 
erythroderma 
 IV- Generalized 
erythroderma with bullous 
formation and/or 
desquamation 
0-  ≤500 mL/day or <280 
mL/m2/day 
 I-  >500 but ≤1000 mL/day or 
280-555 mL/m2/day 
 II-  >1000 but ≤1500 mL/day 
or 556-833 mL/m2/day 
 III-  >1500 mL/day or 833 
mL/m2/day 
 IV-  Severe abdominal pain 
with or without ileus, or stool 
with frank blood or melena 
0-  No protracted 
nausea and vomiting 
 I-  Persistent nausea, 
vomiting, OR biopsy 
showing acute GVHD 
of stomach or 
duodenum 
0-   <2.0 mg/dL 
 I -    2.0-3.0 mg/dL 
 II-    3.1-6.0 mg/dL 
 III-    6.1-15 mg/dL 
 IV-    >15.0 mg/dL   
 
 
Overall Clinical Grading of Severity of Acute GVHD 
Grade 
Skin 
 
Gut 
 
Liver 
 I 
Stage I-II  
& 
None /Stage 0 
& 
None /Stage 0 
 II 
Stage III  
Or 
Stage I  
Or 
Stage I  
 III 
Stage 0-IV  
Or 
Stage II-IV  
Or  
Stage II-III  
 IV 
Stage IV  
Or  
Stage 0-IV  
Or  
Stage IV  
If KPS is ≤ 30%, or decreased ≥ 40% from baseline KPS, the status is Grade IV 
 
 
 
 
 
 

 
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APPENDIX B: CHRONIC GVHD GRADING 
Onset of Chronic GVHD  
*Karnofsky/Lansky score 
at time of diagnosis  
Progressive (acute GVHD progressed directly to chronic) 
Interrupted (acute GVHD resolved, then Chronic developed) 
De novo (acute GVHD never developed) 
Chronic GHVD Flare (symptoms reactivated within 30 days of drug tapering 
or discontinuation) 
Diagnosis 
of 
Chronic 
GVHD based on 
Histologic evidence/biopsy proven 
Clinical Evidence 
Both 
Unknown 
Maximum Chronic GVHD Limited-localized skin involvement and/or hepatic dysfunction due to 
chronic GVHD 
Extensive-generalized skin involvement; or, liver histology showing chronic 
aggressive hepatitis, bridging necrosis or cirrhosis; or involvement of eye: 
Schirmer’s test with <5mm wetting; or, involvement of minor salivary glands 
or oral mucosa demonstrated on labial biopsy; or, involvement of any other 
target organ 
Overall 
Severity 
of 
Chronic GVHD 
Mild-signs and symptoms of chronic GVHD do not interfere substantially 
with function and do not progress once appropriately treated with local 
therapy or standard systemic therapy (corticosteroids and/or cyclosporine or 
FK 506) 
Moderate-signs and symptoms of chronic GVHD interfere somewhat with 
function despite appropriate therapy or are progressive through first line 
systemic therapy (corticosteroids and/or cyclosporine or FK 506) 
Severe-signs and symptoms of chronic GVHD limit function substantially 
despite appropriate therapy or are progressive through second line therapy 
 
 
 

 
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APPENDIX C: KARNOFSKY PERFORMANCE SCALE 
Karnofsky Performance Status 
KPS 100 Normal; no complaints; no evidence of disease 
KPS 90 Able to carry on normal activity; minor signs or symptoms of disease 
KPS 80 Normal activity with effort; some sign or symptoms of disease 
KPS 70 Cares for self; unable to carry on normal activity or do active work 
KPS 60 Requires occasional assistance, but is able to care for most personal needs 
KPS 50 Requires considerable assistance and frequent medical care 
KPS 40 Disabled; requires special care and assistance 
KPS 30 Severely disabled; hospitalization is indicated, although death not imminent 
KPS 20 Very sick; hospitalization necessary; active support treatment is necessary 
KPS 10 Moribund; fatal processes progressing rapidly 
KPS 0 Dead 
 
 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
APPENDIX D: REAGENTS, EQUIPMENT AND SUPPLIES NECESSARY FOR VACCINE PREPARATION 
Reagents 
 
CMV-MVA Triplex vaccine  vials containing 5.1x108  pfu/mL, fill volume approximately 1mL 
(Lot#0786-181-0001-1) OR 9.1x108  pfu/mL, fill volume approximately 1 mL (Lot#0786-181-0002-
1). 
 
PBS containing 7.5% lactose, fill volume 1mL 
 
Equipment 
 
Vortex 
 
Test tube cooling block 
 
NIST thermometer (kept in the IDSA refrigerator) or digital thermometer 
 
microfuge 
 
Supplies 
 
Sterile syringes 1mL and 3mL 
 
Sterile needles 
 
Small sealable amber plastic bag 
 
Alcohol swabs 
 
Container with Ice packs(provided by IDS with prepared dose) 
 
 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
APPENDIX E: SAE/UP REPORTING COVERSHEET 
NOTIFICATION OF UNANTICIPATED PROBLEM/SERIOUS ADVERSE EVENT 
For Use by Participating Institutions Only 
THIS FORM  MUST BE EMAILED TO DCC@COH.ORG WITHIN 24 HOURS OF KNOWLEDGE OF ONSET OF SERIOUS ADVERSE EVENT OR 
UNANCTICIPATED PROBLEM  
COH IRB #14295- Participating Site IRB # ___________ 
 
From: 
Date: 
Phone No.: 
Email: 
 
Reporting Investigator: 
Event:  
Participant ID: 
Institution: 
Date Event Met Reporting Criteria (as defined in protocol):   
Type of Report:  Initial    Follow-up 
Toxicity Grade:   G1/mild    G2/moderate     G3/severe    G4/life threatening    G5 
Attribution to Vaccine:   Unrelated   Unlikely   Possible    Probable   Definite 
Historical/Known Correlation to Vaccine:  Expected    Unexpected   
Meets Definition of Serious AE:      Serious      Non-serious 
Meets Definition of Unanticipated Problem:      UP      Not a UP 
Has the event been reported to the following institution’s IRB?  __ No    __ Yes    Date: __ __ / __ __  / __ __ 
Signature of Investigator:_____________________________Date:______________ 
 
 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
APPENDIX F: REGISTRATION COVERSHEET 
COH IRB 14295: A PHASE II RANDOMIZED, PLACEBO-CONTROLLED TRIAL TO EVALUATE THE PROTECTIVE 
FUNCTION OF AN OPTIMIZED DOSE OF CMV-MVA TRIPLEX VACCINE IN RECIPIENTS OF AN ALLOGENEIC 
HEMATOPOIETIC STEM CELL TRANSPLANT 
 
Data Coordinating Center:  City of Hope 
1500 Duarte Road 
Duarte, CA 91010  
Tel:  626-256-4673 x 3968  
Email:  DCC@coh.org (use #secure# in subject line) 
 
 
 
 
 
 
Principle Investigator:___________________________ 
 
 
Participating Site:_______________________________ 
 
 
 
 
 
 
CRA/Study Coordinator:  _________________________  Contact Number:  ______________________________ 
Patient’s Initials: (F M L): 
Institution: 
Patient's DOB: 
Investigator/Treating Physician: 
Patient’s Zip Code: 
IRB approval valid until (date): 
Sex: 
______Male 
______Female 
Date Informed Consent was Signed: 
Race: 
___ 
Black 
___ 
Caucasian 
___ 
Asian 
___ 
American Indian 
___ 
Native Hawaiian/Pacific Islander 
___ 
Other ____________ 
Ethnicity: 
___ Hispanic 
 
___ Non-Hispanic 
 
___ Other 
______________ 
 
Projected Transplant Date: 
 
Diagnosis: ________________ 
 
 
 
 
 
 
 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
APPENDIX G: DFCI REGISTRATION COVERSHEET 
 
CMV-MVA IN RECIPIENTS OF ALLO HSCT 
 
DANA-FARBER CANCER INSTITUTE/HARVARD CANCER CENTER 
CITY OF HOPE 
 
REGISTRATION COVER SHEET 
 
STUDY INFORMATION 
Sponsor: NIH: NCI: Division of Cancer Prevention and Control 
DFCI IRB #: 15-555 
 
SUBJECT INFORMATION 
Subject Number: ________________ 
Subject initials: _________________ 
Year of Birth: ________________ 
First 3 digits of zip code: _____________ 
Gender: _______________ 
Race: ______________ 
Ethnicity (circle one): 
Hispanic       / 
Non-Hispanic   /   Unknown 
Diagnosis: ______________ 
Date of Consent: ________________ 
Projected HSCT Day 0:______________ 
 

 
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IRB# 14295        Version:  19                                 VERSION DATE:  12/15/2022               
RESEARCH TEAM INFORMATION 
Name of Consenting Investigator: ____________________ 
Name of Registering Person: ________________ 
Contact Phone of Registering Person: __________________ 
Investigator Signature: ______________________     Date:____________________ 
Registering Person Signature: _______________________     Date:____________________