Clinical trial NCT02590068
Conditions: Hepatitis C
Sponsor: Rockefeller University
Start date: 2015-12-01; Completion date: 2017-09-08
Enrollment: 14 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study aims to identify the innate and adaptive immune response to zoster vaccination. Half of the participants will be individuals with chronic hepatitis C, while the other half with healthy volunteers.The innate immune signature elicited by Zoster vaccination will be characterized by RNA-seq analysis of pre- and post-vaccination RNA from whole blood. We will compare fold changes in gene expression profiles pre- versus post-vaccination in each individual, as well as between the two arms of the study. RNA-seq will be used to assess innate immune activation by evaluating the changes to the expression levels of interferon-stimulated genes pre- and post-vaccination. Adaptive immune response will be determined by the traditional correlates of protection used in previous Zoster clinical studies in addition to flow cytometry24. Correlates of protection include antibody response, interferon gamma production and the frequency of responder cells post- vaccination24. For antibody production, we will perform Zoster glycoprotein ELISA (gpELISA) targeting IgG/IgM. The number and frequency of responder cells will be characterized by flow cytometry.

Detailed description:
Chronic HCV infection is associated with persistent innate immune activation and dampened cellular immune responses. Interferons (IFNs) are key mediators of the antiviral innate immune response, initiating the expression of interferon-stimulated genes (ISG) with numerous host protective effector functions. However, in chronic HCV, high pre-therapy expression of ISG and persistent activation of the innate immune system negatively predicts the response to IFN-based therapies and failure of viral clearance8. In addition, HCV persistence is also associated with HCV-specific-CD8+ T cell exhaustion. HCV-specific CD8+ T cell exhaustion is characterized by diminished ex vivo polyfunctionality, upregulation of negative costimulatory cell modulators and, decreased cellular proliferation and IFN production10,12. This phenotype is associated with the development of short-term effector CD8+ T cells rather than durable, long-term memory CD8+ T cells. Chronic bystander infections (e.g. chronic HCV), characterized by persistent inflammation have been linked to bystander (non-HCV specific) CD8+ T cell dysfunction. Bystander CD8+ T cell dysfunction significantly impairs the expansion of memory CD8+ T cells and could prevent the development of secondary immunological memory to new antigens and/or vaccines11,13. Clinically, chronic HCV has been associated with impaired immune response to Hepatitis B vaccination13,14,15. Only 40% to 60% of individuals with chronic HCV achieve seroprotective titers following HBV vaccination versus 90% to 95% in healthy subjects13,14. Specific immune defects responsible for HBV vaccine failure in HCV-infected patients are unknown at present. However, some studies have suggested that the blunting of the immune response to HBV vaccination is associated with lymphocyte dysfunction and upregulation of PD-1 expression on CD4+ T cells in HBV vaccine non-responders13,15.

In the United States, 99.5% of adults over 40 years have been infected with the Varicella zoster virus (VZV) and are at risk of Zoster virus reactivation (shingles) and its complications. Unilateral, painful, blistering rashes along dermatomes characterize shingles. Complications associated with shingles include acute or chronic pain, osteonecrosis, zoster ophthalmicus with visual impairment, increased risk of blindness and a 4-fold risk of cerebral vasculitis-associated stroke)1,2. Overall, complications of shingles have a negative impact on the quality of life and activities of daily living21,22. Zoster vaccine live (Zostavax®, Merck) is recommended for the prevention of shingles. Zoster vaccine is a live, attenuated vaccine that is licensed by the FDA for individuals older than 50 years without an underlying immune deficiency (HIV, malignancies, immunosuppression and transplantation). In non-immunocompromised individuals, Zoster vaccine decreases shingles by 51% in individuals between ages 60 - 89 years and 70% in individuals between 50 - 59 years of age. Chronic infections such as TB, malaria and chronic Hepatitis C virus (HCV) have been associated with increased susceptibility to other pathogens and decreased vaccination efficacy3-6. Although chronic HCV infection is not considered a clinically immunocompromised state, it is associated with persistent immune activation and decreased vaccination response7. Zostavax is routinely administered to chronic HCV patients. However, at present, no other study has documented the immune responses elicited by Zoster vaccination in this population. This study aims to identify the innate and adaptive immune signatures elicited by zoster vaccination in chronic HCV and healthy volunteers. Unrecognized suboptimal vaccine response in individuals with chronic immune dysregulated states (chronic bystander viral infections (HBV, HCV and HIV with CD4 \>200), diabetes, advancing age, cancers and transplantation) could be potentially devastating and costly.

Arms: arm 1: Zoster vaccine live (Zostavax), 0.65 ml dose, administered subcutaneously to Hepatitis C infected volunteers arm 2: Zoster vaccine live (Zostavax), 0.65 ml dose, administered subcutaneously to healthy volunteers
Interventions: intervention 1: Zoster vaccine live
Intervention descriptions: intervention 1: Single Zostavax vaccine, 0.65 ml dose, administered subcutaneously

Protocol documents:
Prot_SAP_000.pdf: 
Title: Effects of Chronic Viral Infection on Immune Response to Zoster Vaccination 
 
NCT Number: NCT02590068 
 
Last Approval Date: 05/15/2019 
 
 


12.0
 
 and Rationale
Objectives
12.1  * Overview
Briefly state the 
. Give enough background and rationale to provide both scientists 
purpose of this study
and lay members of the IRB and ACCTS with the basis for exposing human participants to the risks 
involved.
Zoster vaccine (Zostavax, Merck) is recommended for the prevention of herpes zoster (HZ) reactivation (shingles; painful 
blistering rash along the cranial, thoracic or lumbosacral dermatomes). It is recommended for individuals > 50 years 
without an underlying immune deficiency (HIV, malignancies, immunosuppression and transplantation). In non-
immunocompromised individuals, zoster vaccine decreases HZ reactivation by 51-61%. In the U.S., 99.5% of adults >40 
years have been infected with Varicella zoster virus (VZV) and are at risk of herpes zoster virus reactivation and its 
complications (acute or chronic PHN, osteonecrosis, herpes zoster ophthalmicus with visual impairment, increased risk of 
blindness and a four-fold risk of cerebral vasculitis-associated stroke). Chronic infections with persistent mycobacterial, 
helminthes and viruses are associated with increased susceptibility to other pathogens and decreased vaccination 
efficacy. Although chronic Hepatitis C virus (HCV) infection is not considered a clinically immunosuppressed state, it has 
been associated with persistent immune activation and decreased immune response to vaccination. Zostavax is routinely 
administered to chronic HCV patients. However, at present, no other study has documented the immune responses 
elicited by Zostavax in this population. This study aims to identify the innate and adaptive immune signatures elicited by 
zoster vaccination in individuals chronically infected with HCV as compared to healthy volunteers. Methods to be Results 
from this study could identify targets of potential vaccine optimization for this population and increase available knowledge 
on host immune response to chronic viral infections.
12.2  
 Engaging Stakeholders: Describe any plans to engage other stakeholders (Scientists, practitioners, 
*
patients, advocacy groups, etc.) for hypothesis generation, or feasibility purposes.    
The PI along with other members of the Rice laboratory were instrumental in the design of this study. 
Biostatisticians at the Rockefeller University provided additional statistical evaluations for the Clinical and 
Translational Science Center.
The team will identify multiple members of the patient community, as well as outreach and harm-
reduction workers with experience referring HCV infected volunteers to clinical research, in order to 
engage patients in the design of the study, including aspects of recruitment, conduct, retention, and the 
analysis and sharing of results. Patients and other will be invited to face-to-face meetings with the 
research team and support staff (or teleconference if preferred), and will be compensated for their time
/effort.  Patient stakeholders engaged in this way are not expected to enroll into the study; any 
exploration of their enrollment would be made separately, free of undue influence
12.3  * Hypothesis
Describe the 
 in a single sentence.
research hypothesis
We hypothesize that individuals with chronic HCV infection will demonstrate impaired innate and adaptive 
immune responses following zoster vaccination.
12.4  
Aim(s)
 
*


Indicate how you will 
 (e.g., to compare groups, to estimate a parameter, to 
address the hypothesis
ascertain feasibility). Since the sample size determination is usually based on the primary aim only, the 
primary aim should be sufficient to justify the study.
AIM 1: Characterize the adaptive immune response to zoster vaccination in chronic HCV 
infection
Zoster vaccine will be administered to healthy and HCV-infected individuals. Volunteers serum, PBMCs, 
and plasma will be collected at screening, days 0, 3, 7, 14, and 42 and batched for analysis to limit 
variations. In order to fully evaluate the immune signature generated by the zoster vaccine, we will 
determine the frequency and functionality of specific PBMC populations using flow cytometry and ELISpot 
assays. We aim to compare the data generated from this study to other published human system 
immunology and/or vaccination studies. To achieve this aim,  our research group has adapted a flow 
cytometry panel designed to limit variations in standardizing human immunophenotyping. This flow 
cytometry panel is also currently being applied to the HBV vaccine study (CRI-0844).  We will analyze 
intracellular cytokine staining and expression of cell surface markers on immune cells. IFN-gamma 
ELISpot assay has been used in older HZ vaccine studies to evaluate T-cell immune response post-
vaccination. Commercially available VZV-derived peptides pools will be used to stimulate PBMCs in the 
ELISpot assay. VZV-specific antibody production post-immunization would also be assess using 
commercially available VZV-ELISA kits.
AIM 2. Characterize the innate immune response to zoster vaccination by RNA-Seq analysis
RNA-seq will be used to evaluate innate immune activation by evaluating the changes to the expression 
levels of interferon-stimulated genes pre- and post-vaccination. In this pilot project, RNA will be isolated 
with using commercially available PAXgene tubes to immediately stabilize intracellular RNA profile and 
traditional homogenization of isolated volunteer PBMCs in Trizol prior to cryopreservation. RNA-seq 
sample preparation and sequencing will be performed at the Rockefeller University Genomics Resource 
center. The Rosenberg research laboratory, with their extensive experience with RNA-seq will perform 
analysis of this data. Genome-wide mRNA expression profiles will be compared between the 2 arms of the 
study, healthy and chronically HCV infected volunteers. Gene expression levels will be quantified in 
relation to a reference standard of External RNA Control Consortium (ERCC) controls – a calibrated panel 
of defined RNA molecules at defined concentrations that will be “spiked in” to each library preparation to 
serve as a universal reference across samples.
12.5  * Primary Outcome(s)
Indicate which 
 will be assessed to judge the primary specific aim.  Give measurement units, 
variable(s)
if applicable.
We will measure the following parameter:
 
- 
antibody levels by gpELISA expressed in (units/ml)
Serum zoster 
 
12.6  * Secondary Outcome(s)
Indicate which 
 will be assessed to judge the secondary outcome(s). Give 
additional variable(s)
measurement units, if applicable.
 
- 
expressed as fold-
Interferon-stimulated gene (ISG) expression in PBMCs 
change (FCH) above baseline
 
-
(expressed in %)
 Serum biomarkers of innate immune activation 
 
- 
 VZV specific CD4+ T cells post-vaccination 
Responder cell frequency:
(expressed in %)
 
-
 by ELIspot expressed as number of spot-forming 
 Interferon-gamma response
cells per 10  PBMCs
6
 


12.7  * Methods and Procedures
Please provide a description of the laboratory and clinical analyses and procedures that will be performed. 
Include the role of external collaborators and consultants when appropriate. Please refer to Help text for 
Guidance.
This study is an open-label protocol divided into the healthy versus chronic HCV 
volunteer arms. The study will be advertised in local newspapers and different 
online resources. Potential participants identified with the aid of the Bellevue 
Hospital Center Hepatitis clinic and other local physicians, will be asked to call 1-
800-RUCARES (1-800-782-2737), if they  are interested in the study.
Our aim is to screen and enroll 20 age-matched (+/- 5 years) and gender-matched 
individuals between 50 and 60 years of age into each arm of the study. Individuals 
recruited into the study must be HIV and hepatitis B negative, without a history of 
vaccination within the past month, no history of immunosuppressive therapy, active 
malignancies or herpes zoster reactivation. Baseline screening tests (HIV ELISA, 
hepatitis B anti-surface and core antibodies, hepatitis C antibody, viral load and 
genotyping, complete blood counts and a comprehensive metabolic panel) will be 
obtained prior to enrolling in the study. HCV viral load and genotyping: will be 
determined by clinical laboratory analysis. Patient blood samples will be sent to a 
CLIA certified reference laboratory for analysis.
The initial protocol was written in the summer of 2015, prior to the availability of the 
influenza vaccine for the current 2015 – 2016 season. It would be unethical to have 
interested participants delay receipt of their influenza vaccination prior to enrolling 
in this study. Also, a 3-month delay after participants’ influenza vaccination would 
delay the progress of the clinical study, as we have already secured some funding 
for the study.
In addition, receipt of vaccination 4 weeks prior to entry into a zoster vaccination 
study has a precedent, namely the landmark study by Oxman et al.  The FDA’s 
approval of a license for use of Zostavax in 2006 cited this study .
1
Reference:
1.
Oxman, M. N.
 A vaccine to prevent herpes zoster and postherpetic
 et al.
neuralgia in older adults. N Engl J Med. 2005 Jun 2;352(22):2271-84.
In this study there will be a total of seven study visits (Screen I and II, Days 0, 3, 7, 
14, 42).  All enrolled volunteers will be given a single 0.65 mL dose of the live 
attenuated zoster vaccine (Zostavax, Merck & Co, Inc) on day 0 with follow-up 
visits on days 3, 7, 14, and 42. The most frequent adverse reactions, reported in 
subjects vaccinated with Zoster vaccine, were headache and injection-site 
reactions. Additional adverse effects have not been reported in individuals 
chronically infected with Hepatitis C virus.  All women of child-bearing potential are 
required to use a highly effective form of contraception such as condoms, birth 
control pills, birth control injections, or intrauterine devices for three months 
following the vaccination.  Any adverse reactions observed in women of child-
bearing potential should be reported to:  To report SUSPECTED ADVERSE 
REACTIONS or exposure during pregnancy or within three months prior to 
conception, contact Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., 
at 1-877-888-4231 or VAERS at 1-800-822-7967 or www.vaers.hhs.gov.


At the Day 0 and Week 6 visit we will collect approximately 121 mL of blood during 
and at the other visits (Days 3, 7, and 14) we will collect 49 or 53 ml of blood 
repectively for research and clinical laboratory monitoring. Non-fasting blood 
samples will be drawn during the initial screening visit. Subsequent laboratory 
blood samples will be fasting for screening visit II and days 0, 3, 7, 14 and 
42. Research blood will be separated into PBMCs, plasma and serum by the study
PI. All samples will be stored in cryopreserved aliquots until batched assays prior to 
described assays.
HCV persistence has been associated with HCV-specific-CD8+T-cell exhaustion 
and bystander CD8+T-cell dysfunction. HCV-specific CD8+T-cell exhaustion is 
characterized by diminished 
 polyfunctionality, upregulation of negative co-
ex vivo
stimulatory cell modulators, decreased cellular proliferation, inteferon production 
and cytotoxicity. Effective immune responses to vaccination include the activation 
and expansion of antigen-specific memory B-cells and T-cells following antigen 
stimulation. Chronic HCV has been associated with impaired immune response to 
vaccination against Hepatitis A virus, influenza and most prominently Hepatitis B 
virus with 40-60% in chronic HCV versus 90-95% in healthy volunteers. Studies on 
HBV-vaccination of chronic HCV patients have identified impaired specific and non-
specific CD4+T-cell immune in non-responders to HCV infection.
Flow cytometry, ELISpot and ELISA analyses will be performed on PBMCs to 
determine B-cell and T-cell proliferation and polyfunctionality. we will determine the 
frequency and function of specific PBMC populations using flow cytometry and 
ELISpot assays. For flow cytometry, we will utilize an antibody panel designed to 
limit variations in standardizing human immunophenotyping to aseess intracellular 
cytokine staining and expression of cell surface markers on immune cells. IFN-
gamma ELISpot assay has been used in older HZ vaccine studies to evaluate T-
cell immune response post-vaccination. We will utilize commercially available VZV-
derived peptide pools will be used to stimulate PBMCs in the ELISpot assay. VZV-
specific antibody production post-immunization would also be assess using commer
cially available VZV-ELISA kits. A multiplex Luminex assay targeting TNF-alpha, 
IFN-gamma, IL-2, IL-4, IL-17 and other cytokines will be performed on plasma 
samples. Flow cytometry and multiplex cytokine assays will be performed with the 
assistance of the Translational Technology Core Laboratory Resource center.
Chronic HCV infection is associated with persistent innate immune activation and 
dampened cellular immune responses. Interferons (IFNs) are key mediators of the 
antiviral innate immune response, initiating expression of interferon-stimulated 
genes (ISGs) with numerous host protective effector functions. In chronic HCV, 
high pre-therapy expression of ISGs and persistent activation of the innate immune 
system negatively predict the response to IFN-based therapies and failure of viral 
clearance. Innate immune response will be determined by RNA-seq pre- and post-
vaccination. RNA will be isolated using commercially available PAXgene tubes to 
immediately stabilize intracellular RNA profile, in addtition to the traditional homogen
ization of isolated volunteer PBMCs in Trizol prior to cryopreservation. Blood will be 
collected into PAXgene tubes and isolated PBMCs will also be collected into TRIzol 
RNA isolated from PBMC will be prepared 
for RNA isolation and RNA-seq analysis. 
by the Genomic Resource center and sequenced for genome-wide mRNA 
expression profiling. Genome-wide mRNA expression profiles will be compared 
between the two arms of the study (healthy and chronically HCV infected 
volunteers). Gene expression levels will be quantified in relation to a reference stan
dard of External RNA Control Consortium (ERCC) controls – a calibrated panel of 
defined RNA molecules at  defined concentrations that will be “spiked in” to each 
library preparation to serve as a universal reference across samples.


PBMC samples may also be analyzed by single cell resolution InDrop RNA-Seq 
(REFERENCE PMID: 26000487), established for immune response profiling in the 
Rosenberg laboratory.  Within a microfluidic device, individual cells and individual 
hydrogel “barcode beads” are co-encapsulated in water-in-oil droplets, in which 
reverse transcription tags nascent cDNA with cell-specific barcode sequences. 
After droplet breakage, library preparation and high throughput sequencing, reads 
can be assigned to individual cells by barcode for gene expression profiling. Using 
this approach, sufficient sampling can be achieved with relatively low input cell 
numbers and rare cells (i.e. dendritic cells, antigen-specific lymphocytes) are 
represented in resulting datasets.
 
In order to define the immune functions engaged by Zoster vaccination at single 
cell resolution, longitudinal PBMC samples will be analyzed by InDrop RNA-
Seq.  Data analysis will focus on first classifying cells by type (e.g. T cells, B cells, 
NK cells, classical monocytes, dendritic cells, etc.) using multidimensional 
clustering methods as implemented in the Seurat tool (REFERENCE PMID: 
25867923). Next, differential gene expression patterns active in different cell 
subsets will be assessed over time, and the heterogeneity of these patterns will be 
evaluated at single cell resolution. We expect these results to complement bulk 
RNA-Seq results by providing greater detail on specific cell subsets and their 
respective transcriptional networks and response dynamics.
 
Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, et al. Droplet 
Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells. Cell. 
Elsevier; 2015;161: 1187–1201. doi:10.1016/j.cell.2015.04.044
 
Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of 
single-cell gene expression data. Nat Biotechnol. 2015. doi:10.1038/nbt.3192.
 
With the May 2019 IRB continuing review submission, Dr. Jegede has been 
replaced by Dr. Rice as the Principal Investigator of this study.  Dr. Jegede has 
been listed as External Personnel and will not conduct any further data analysis or 
be involved with any protocol-related activities. 
 
With the May 13, 2019 amendment, Dr. Rosenberg, a Guest Investigator in the 
Rice laboratory, will conduct the data analysis and review of the stored research 
samples collected in this study.
 
12.8  
 
* Data Analysis
Describe method(s) of data analysis.  Include the role of external collaborators as appropriate.
The primary analysis for the set of primary outcomes will search for statistical evidence of impairment in 
the innate and adaptive immune response in chronic-HCV subjects compared to age-matched Healthy 
Controls by testing the hypotheses that the variation from baseline to day 42 will differ between these two 
groups.  To achieve this goal, we will apply two-sided paired t-tests and/or Wilcoxon tests for each one of 
the outcomes. Additionally, as a secondary analysis, we will fit a mixed-effects model that will use group 
(HCV – HC) and time (0,3,7,14, and 42 days) as fixed effects and a random intercept for each subject. 
This modeling strategy allows to infer about differences across time points, and also within time points, 
while controlling for multiple testing.  Further evaluation of the immune signature generated by the zoster 
vaccine will be done with flow cytometry and ELISpot assays and qualitative comparisons with other 


human system immunology and /or vaccination studies will be carried out. For the secondary outcomes, 
the analysis for the ISG expressions as measured by RNA-seq counts will be processed in 
 package 
limma
in R and the same formulation for mixed-effects model described previously will be applied to verify 
differences at gene-wise level. Serum biomarkers of immune activation will be compared across groups 
with paired t-tests and Wilcoxon test for each time point.
12.9  
 
* Explain the rationale for the choice of statistical measures and the number of participants 
proposed for the study, including the power calculations when applicable.
A sample of n=40 (20 HCV / 20 HC) individuals provides 80% power at 1.6% (after Bonferroni correction 
for 3 outcomes) significance to detect minimum differences in biomarkers variation from baseline to day 
42 as 1.08 standard deviations when applying two-sided paired t-tests. Since there is no preliminary data 
showing the degree of variability in the primary outcomes for the chronic HCV population, this pilot study 
will be valuable for future power analysis. To test for gene-wise differences between healthy and HCV-
infected groups for a single time point, a sample size of n=20 individuals in each groups provides 80% 
power at 5% significance to detect effect sizes greater than 1.5 FCH (Fold-Changes), assuming a 
coefficient of variation as 40% and a depth of coverage greater than 20. Calculation was performed using 
the library RNASeqPower in R software version 3.2.1.
12.10  * Will samples be coded?
 Yes   
 No
If Yes, Please describe coding scheme consistent with GCP.  If samples will not be coded, please provide 
justification for this proposed departure from GCP practice.
All samples will be coded with unique identifiers based on study number assigned 
to each individual upon study enrollment. Study volunteers' HIPAA protected 
information will not be associated with any research data. All computers used to 
store and analyze research data will be password protected. All data will be backed-
up into the system maintained by Information Technology Service at the 
Rockefeller University.
If available, upload the Data and Sample Sharing Management Plan approved by RU IT.
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