Host Mechanisms Involved in Achieving SVR Using Grazoprevir and Elbasvir in Treatment of Chronic Hepatitis C in Patients With CKD Before and After Renal Transplantation
Phase: PHASE4
Conditions: Hepatitis C, Renal Insufficiency, Chronic, Disorder of Transplanted Kidney
Sponsor: University of Maryland, Baltimore

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Section #1 - MISP Protocol Identification 
 
Study Title: 
  
Host mechanisms involved in achieving SVR using a fixed dose combination of 
grazoprevir and elbasvir in treatment of chronic hepatitis C genotype 1 in non-
cirrhotic patients with chronic kidney disease after renal transplantation. 
 
Version Date: 
 
June 8, 2018 
 
Institution Name 
 
University of Maryland School of Medicine, Institute for Human Virology 
 
Investigator Contact 
Information: 
 
Principal Investigator:  Jennifer Husson MD, MPH 
University of Maryland School of Medicine, Institute for Human Virology 
725 W Lombard St, N147 
Baltimore, MD 21201 
T: 410-706-6973 
F: 410-706-3243 
jhusson@ihv.umaryland.edu 
 
Co-Investigator: Shyamasundaran Kottilil, MBBS, PhD 
725 W Lombard St, S222 
Baltimore, MD 21201 
T: 410-706-8927 
F: 410-706-3243 
skottilil@ihv.umaryland.edu 
 
Co-Investigator:  Abdolreza Haririan MD, MPH 
655 W Baltimore St, 217A 
Baltimore, MD 21201 
T: 410-706-2842 
F: 410-328-5685 
ahariria@medicine.umaryland.edu 
 
Study Coordinator:  Ilise Marrazzo, RN MPH 
725 W Lombard St, N142 
Baltimore, MD 21201 
T: 410-706-2564 
F: 410-706-3243 
IMarrazzo@ihv.umaryland.edu 
 
 
 
 

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Section #2- Core Protocol 
 
2.1 Objectives 
& Hypotheses 
2.1 Objectives and Aims  
 
Aims 
1. To evaluate the determinants of Sustained Virologic Response (SVR) in patients 
treated with grazoprevir and elbasvir in the presence of immunosuppression (post-
renal transplant). 
 
Primary Objective 
1. To determine the difference in the host immune response as measured by 
interferon stimulated gene (ISG) expression (IP-10 in particular), and HCV specific 
T-cell response in post-renal transplant patients compared to historical, non-
transplant patients and its association with SVR. 
2. To examine the efficacy of grazoprevir and elbasvir in patients post- renal 
transplantation compared to historical, non-transplant patients. 
 
Secondary Objectives  
1. To evaluate the safety and adverse events of grazoprevir and elbasvir post-renal 
transplant patients compared to historical, non-transplant patients.  
2. To describe the effects of chronic HCV treatment on two-year renal allograft 
outcomes in patients treated after renal transplantation.  
 
2.1.1 Clinical hypotheses  
 
Primary Hypotheses 
1. Patients treated for chronic HCV with grazoprevir and elbasvir after renal 
transplantation will maintain a robust host immune response as measured by ISG 
expression (IP-10 in particular) and HCV specific T cell responses in the presence 
of immunosuppression. 
2. The proportion of patients who achieve SVR12 will be greater than 90% in post-
renal transplant patients. 
 
Secondary Hypotheses 
1. The safety, tolerability, and adverse events of treatment with grazoprevir and 
elbasvir in patients with chronic kidney disease will not be significantly different in 
post-transplant patients compared to non-transplant patients. 
2. The treatment of chronic HCV using grazoprevir and elbasvir will improve the renal 
allograft outcomes in terms of rejection or graft survival. 
 
2.1.2 Clinical Impact 
 
Chronic HCV in patients with CKD is associated with an increased risk of death as well as 
renal transplant graft failures (1).  With the FDA approval of grazoprevir and elbasvir, we 
now have a regimen that can be used in these patients with severe CKD.  However, it 
remains unknown if there is an immunologic advantage for both achievement of SVR 12 
and renal allograft outcomes in treating these patients prior to their transplant compared to 
waiting until after transplantation when they will be immunosuppressed. And while we 
predict that transplant recipients will maintain high rates of SVR, it is uncertain whether 
these immunosuppressed transplant recipients will maintain robust host immune responses 
to HCV treatment. 
 

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2.2 
Background & 
Rationale, 
Significance 
of Selected 
Topic & 
Preliminary 
Data  
 
 
Background 
 
Hepatitis therapy has evolved rapidly from an interferon based, prolonged regimen with 
moderate efficacy to oral, direct acting antiviral (DAA) based regimens with high efficacy. 
Recently, sofosbuvir/ledipasvir, as single tablet combination and Viekira pak, a combination 
pill of multiple DAAs, have all been approved for the treatment of hepatitis C in genotype 1 
and 4 infected patients. However, limited data exist in the use of either of these regimens in 
patients with renal disease, particularly those with CKD stages 4 and 5.  
 
Sofosbuvir enters the hepatocyte where it is metabolized to its active form, GS-461203. 
The downstream inactive nucleoside metabolite GS-331007 is almost exclusively 
eliminated from the body renally, mediated through a combination of glomerular filtration 
and active tubular secretion (2). Results of phase II and III clinical trials of sofosbuvir have 
previously excluded patients with serum creatinine levels greater than 2.5 mg/dL or 
creatinine clearance (CrCl) level less than 60 mL/min. In subjects with end stage renal 
disease (ESRD) (relative to subjects with normal renal function), sofosbuvir and GS-
331007 AUC (0-inf) were 28% higher and 1280% higher, respectively, when sofosbuvir was 
dosed 1 hour before hemodialysis compared with 60% higher and 2070% higher, 
respectively, when sofosbuvir was dosed 1 hour after hemodialysis. No dosage adjustment 
is required for patients with mild or moderate renal impairment (CrCl 30 mL/min-80 mL/min) 
(2). Thus, the safety of sofosbuvir has not been established in patients with severe renal 
impairment (CrCl <30) or ESRD. Unlike with sofosbuvir, no clinically relevant changes in 
ledipasvir pharmacokinetics were found between volunteers with normal renal function and 
those with severe renal impairment (eGFR <30 mL/min by Cockcroft-Gault) after a single 
dose of 90 mg of ledipasvir was administered (3). 
 
HCV-TARGET is an ongoing prospective observational cohort study characterizing the use 
of DAA agents across clinical practices in North America and Europe. The study reported 
adverse events and efficacy of sofosbuvir containing HCV treatment regimens in patients 
with variable degree of renal dysfunction (eGFR <30, 31-45, 46-60 and >60). The patients 
received different regimens that included sofosbuvir (peg/sof/rbv; sof+ sim ± rbv, sof/rbv). 
Overall, the regimens were well tolerated with no increased discontinuation among patients 
with low eGFR. The SVR12 rates were similar across the groups regardless of renal 
function. Notably, there were progressive deterioration of renal function and renal 
symptoms in the eGFR <30 ml/min suggesting the need for close monitoring of these 
patients. In summary, patients with low baseline renal function have a higher frequency of 
anemia, worsening renal dysfunction and SAEs, while the treatment responses remain high 
and comparable to those without renal impairment (4). 
 
Viekira Pak consists of paritaprevir/ritonavir, ombitasvir and dasabuvir, all cleared by 
hepatic metabolism. Single dose pharmacokinetics of paritaprevir/ritonavir, ombitasvir and 
dasabuvir was evaluated in HCV negative volunteers with mild (eGFR 60-89 ml/min), 
moderate (eGFR 30-59ml/min) and severe (<30ml/min) renal dysfunction. The results 
concluded that changes in the pharmacokinetics were not considered to be clinically 
relevant in HCV infected patients (5).  Thus, Viekira Pak remains an option for some 
patients with CKD.  However, there are significant drug-drug interactions between the 
ritonavir in Viekira Pak, which is a CYP3A inhibitor and tacrolimus, which is a substrate of 
CYP3A and which a majority of renal transplant patients are on post-transplantation. 
 
Twenty patients with HCV genotype 1 infection and CKD stages 4/5 (eGFR <30 without 
cirrhosis) were treated with AbbVie 3D ± RBV in a multicenter open-label phase IIb study. 
Notably, 70% of patients were African Americans and 65% had CKD receiving 
hemodialysis. Ribavirin (GT1a only) was dosed 4 hours before hemodialysis and monitored 
with weekly hemoglobin assessments. Though still ongoing, all patients thus far have 
achieved SVR4 (10/10) and SVR12 (2/2). Interestingly, 8 of 13 patients required 

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interruption of RBV due to a drop in hemoglobin and 4 of 8 patients also required 
erythropoietin during the first 7 weeks of therapy. Mean drug concentration (Ctrough) of all 
drugs were measured which were within the range that was observed with previous 
pharmacokinetic studies in healthy volunteers (6). In summary, HCV genotype 1 non-
cirrhotic patients treated with AbbVie 3D +/- RBV can result in viral suppression in most 
patients. However, increased frequency of ribavirin–induced anemia can occur frequently, 
requiring close monitoring of all patients and judicious dose reductions of RBV. In addition, 
the use of this ritonavir containing regimen is limited in the renal transplant population due 
to drug-drug interactions with calcineurin inhibitors. 
 
Recently, the combination of elbasvir/grazoprevir has shown efficacy in both treatment 
naïve and null responders with chronic HCV genotype 1 infection with or without cirrhosis 
(7).  Both grazoprevir and elbasvir are cleared by hepatic metabolism and hence, could be 
used in patients with impaired renal function, including renal transplant candidates or 
recipients.  Grazoprevir is a weak inhibitor of CYP3A4 and thus the area under the curve 
for single-dose tacrolimus increased by approximately 40%, but decreased the Cmax by 
approximately 40% (8). 
 
Evaluation of host immune response to treatment 
 
Persistent activation of host inflammation in the liver is partially mediated by endogenous 
interferons, which contributes to the pathologic development of hepatic fibrosis (9).  This 
activated response is present in both acute and chronic infection; however, it is unable to 
eradicate HCV (10). Increased mRNA expression of these ISGs in liver biopsy samples is 
associated with failure of interferon based therapies (11).  It has been previously 
demonstrated that host responses play an important role in achieving SVR in patients with 
chronic hepatitis C undergoing therapy using DAAs. Clearance of HCV during DAA 
treatment is associated with a downregulation of type II and III IFNs along with their 
respective receptors and ISGs and SVR was associated with an increased ISG expression 
in the liver and IFNA2, which could play a role in eliminating residual HCV (11). 
 
IP-10, an interferon gamma inducible CXC chemokine targeting T lymphocytes and NK 
cells and monocytes, has been shown to be produced by hepatocytes. Elevated pre-
treatment IP-10 levels have been associated with a poorer response to therapy and inability 
to achieve SVR (12). 
 
In addition to the ISG expression mediated response, T cells also play a role in viral 
clearance.  Enhanced polyclonal CD4+ T cell responses have been found in cleared acute 
HCV infections and this response persists in those who permanently clear infection (13) 
(14).  In cleared chronic infection, polyclonal and persistent CD4 T-cell responses directed 
at a variety of different HCV-specific proteins have similarly been detected (13). 
 
Rationale for the study 
 
Host immunity is also likely a major determinant of achieving SVR renal transplant 
recipients with variable degree of immunosuppression. However, it is unclear what role 
immunosuppression may play in modulating this host immune response.  The modulation 
of this response by immunosuppression in patients who are post-transplantation could have 
significant implications for clinical outcomes and achievement of SVR as well as the 
maintenance of SVR over time.  In this regard, we would like to evaluate host response to 
DAA therapy using grazoprevir and elbasvir in patients with CKD by evaluating three 
previously well characterized host responses associated with favorable SVR outcomes.  
These include ISG expression (11), IP-10 levels (15) and HCV specific T-cell responses 
(16).  
 

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A recent study (C-SURFER) evaluated the safety and efficacy of 12 weeks of grazoprevir 
and elbasvir for HCV genotype 1 patients with CKD stages 4/5. The study was designed to 
randomize eligible patients to either immediate or deferred treatment with grazoprevir and 
elbasvir. The delayed treatment arm received placebo and was treated with grazoprevir 
and elbasvir subsequently. The study participants were HCV genotype 1, CKD stages 4/5 
(eGFR <30), and 76% were on hemodialysis, and 46% were African Americans.  A small 
number of patients with compensated cirrhosis were allowed. The study reported an 
intention to treat (ITT) and modified ITT analysis of 94% and 99% for SVR12. There were 
no changes in hemoglobin or other adverse events or erythropoietin use in the immediate 
treatment group compared to placebo.  Four percent of patients in the deferred treatment 
group discontinued treatment due to an adverse event, mainly headache, nausea or fatigue 
(17). In summary, a regimen of grazoprevir and elbasvir could be an effective regimen to 
treat HCV genotype 1 infection in patients with severely compromised renal function.  
 
While studies evaluating the safety and efficacy of this regimen in patients with chronic 
kidney disease have shown success with few adverse events, little is known about the 
efficacy of this, or any other, therapy in patients on immunosuppression after renal 
transplantation or whether it is optimal to treat hepatitis c before or after transplantation.  
Because waiting until after transplant to treat patients for HCV is advantageous by allowing 
those patients to receive HCV-positive donor organs, most centers are waiting to treat 
these patients.  In order to observe whether there is any difference immunologically or 
clinically in treating patients before or after renal transplantation, we will compare our 
transplant cohort to a cohort of historical, non-transplant patients treated for HCV in whom 
we have stored blood samples from prior IRB-approved studies. 
 
Significance  
 
The prevalence of chronic hepatitis C in patients with CKD is much higher than that 
observed in the general population (18). This highlights the importance of finding a ribavirin 
free regimen that is not excreted through the kidneys.  However, it is unclear what the 
timing of HCV therapy should be for these patients.  
 
Additionally, the effect of immunosuppression on the host response to HCV therapy is 
unknown.  On the one hand, these patients could be treated for hepatitis C infection prior to 
renal transplantation to avoid the impact of immunosuppression as well as the impact of 
HCV-related liver and kidney disease on allograft function post transplantation. On the 
other hand, this approach will eliminate access to HCV positive kidneys (making the wait 
list time longer) and potentially reduce the efficacy of DAA therapy (due to drug-drug 
interaction between calcineurin inhibitors and HCV drugs). Recent studies have shown that 
grazoprevir is a weak inhibitor of CYP3A4 and may increase tacrolimus levels (approx. 
40% above expected), though change has not been seen in elbasvir or grazoprevir levels 
(19) (8).  
 
There are real benefits in waiting to treat HCV in patients until after transplantation in that it 
could decrease the wait time for transplantation by not necessitating a delay in transplant 
while completing HCV therapy and by permitting the use of HCV-positive kidney donors.  
Showing the impact that immunosuppression has on the host immune response to HCV 
during therapy would enable clinicians to decide whether patients should be treated before 
or after transplant, especially in cases where the wait for a transplant may be quite long.   
2.3 Study 
Design 
 
Experimental design 
 
The study will be a pilot, prospective, single-center, open-label, non-randomized, non-
controlled clinical trial.  25 HCV genotype 1 infected post-renal transplant patients will be 
enrolled in the study.  Recruitment will be conducted through the renal transplant and 
nephrology outpatient clinics at the University of Maryland. 
 

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Study population 
 
The study will involve a single cohort of post-renal transplant patients. 
 
All patients will have HCV genotype 1 chronic infections with active viremia and have no 
cirrhosis at the time of enrollment.  All patients will additionally have stable renal function. 
 
The post-transplant patients will include renal transplant recipients of both living donor and 
deceased donor organs infected with HCV prior to their transplantation with stable GFRs 
and active HCV viremia.  These patients will also be recruited from the University of 
Maryland’s multidisciplinary transplant nephrology clinic. 
 
Inclusion criteria 
 
 
At least 18 years of age at the time of screening 
 
Have stable renal function for one month (30 days) prior to enrollment 
 
Have Chronic HCV infection prior to transplantation 
o 
Documented HCV viremia ≥ 1,000 IU/ml at screening 
o 
Either documented HCV Ab positivity or HCV viremia ≥ 1,000 IU/ml at least 
6 months prior to enrollment 
 
Documented genotype 1 HCV infection prior to enrollment and after their transplant 
in the post-transplantation cohort 
 
HCV disease staging within 12 months prior to enrollment by liver biopsy, transient 
elastography, or biochemical testing 
 
Be able to give informed consent and comply with study guidelines 
 
Women of childbearing age will be required to have a negative pregnancy test at 
enrollment and use birth control throughout the duration of treatment. 
 
Patients will have undergone renal transplantation no greater than five years prior 
to enrollment, and will be followed in our University’s nephrology and infectious 
disease clinic.  They will all have stable renal function at the time of enrollment. 
 
Exclusion criteria 
 
 
Documented positive HBsAg, and/or HBV DNA prior to enrollment 
 
Any prior exposure to HCV protease inhibitor therapy 
 
HIV co-infection if on a protease inhibitor, etravirine or efavirenz based regimen or 
elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate 
 
Increase in creatinine of 15% or greater within one month (30 days) of the 
screening visit 
 
Evidence of HCC at the time of enrollment 
 
Liver disease caused by an etiology other than HCV 
 
F4 or decompensated cirrhotic patients 
 
Child Pugh class B or C 
 
AST or ALT >350 within 6 months prior to enrollment 
 
Albumin < 3g/dL at the time of enrollment 
 
Platelet count < 75,000 at the time of enrollment 
 
History of clinically significant allergy or adverse event with protease inhibitors 
 
Evidence of the acquisition of HCV at the time of or after transplantation 
 
Pregnant or breastfeeding women 
 
Cyclosporine; St. John’s Wort; Efavirenz;, Phenytoin; Carbamazepine; Bosentan; 
HIV Protease Inhibitors; modafinil;  Ketoconazole; or Rifampin use within 7 days of 
enrollment 
 
Coadministration of more than 20 mg atorvastatin; 10 mg rosuvastatin; 20 mg of 
fluvastatin, lovastatin or simvastatin 
 

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2.4 Study 
Flowchart 
 
Patient screening and enrollment 
 
Figure 1. Study Design 
 
 
 
 
 
 
 
 
 
Schedule of lab work 
A full list is provided as Appendix 1. 
 
Table 1: Schedule of labs by week (wk) 
 
Screen 
Day 
0 
Wk 
1 
Wk 
2 
Wk 
4 
Wk 
8 
Wk 
12/16* 
Post-
Wk 1 
Post-
Wk 2+ 
Post-
Wk 4 
Post-
Wk 12 
Exam 
X 
X 
 
 
X 
 
X 
 
 
 
X 
CBC/CMP 
X 
X 
 
X 
X 
X 
X 
 
 
X 
X 
HCV VL 
X 
 
 
 
X 
 
X 
 
 
 
X 
Tacrolimus 
 
X 
X 
X 
X 
X 
X 
X 
X+ 
X 
 
ELISPOT (PBMCs) 
 
X 
 
 
 
 
 
 
 
 
X 
ELISA 
 
X 
 
 
 
 
 
 
 
X 
 
Paxgene DNA 
 
X 
 
 
 
 
 
 
 
 
 
Paxgene RNA  
 
X 
 
 
 
 
 
 
 
X 
 
Stored Samples 
(Serum, Plasma, 
PBMCs) 
 
X 
 
 
 
 
 
 
 
X 
 
*Wk 16 will be completed instead of week 12 only for those on 16 weeks of therapy. 
+ Post-Treatment Wk 2 will only be done if clinically indicated based upon Post-Tx Week 1 
labs. 
 

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RAV testing (for NS5A positions 28, 30, 31 or 93) will be performed for all patients with HCV 
genotype 1a infection at the time of enrollment as per clinical recommendations (20). 
2.5 Study 
Procedures 
 
Subject identification 
Patients will be identified for screening through the renal transplant clinic or nephrology 
clinics at the University of Maryland. 
 
2.5.1 HCV Treatment Arm  
 
Screening 
All patients will be screened at the IHV Clinical Trials Unit infectious disease, or renal 
transplant outpatient clinic at the University of Maryland. All patients will sign an informed 
consent as approved by our Institutional Review Board (IRB) prior to study participation. At 
this visit, all patients will have clinical and research screening labs drawn and a history and 
physical examination performed.  Additional requirements will be genotype testing prior to 
enrollment, but after transplant for the post-transplantation cohort, and disease staging 
within 12 months of enrollment by liver biopsy, elastography, or biochemical testing.  For 
those who do not have a genotype or disease staging within the specified time frame, 
genotyping and elastography will be repeated as part of the study screening work up.  
Eligibility will be determined based upon these results within 6 weeks of starting the study 
drugs. 
 
Treatment-naïve or treatment-experienced patients who failed prior therapy with pegylated 
interferon and ribavirin have a 5-10% incidence of having baseline resistance associated 
mutants (RAVs). Given the reduced efficacy of this regimen in patients with genotype 1a 
with the presence of baseline NS5A RAVs (58-91% depending upon the methodology vs. 
100% for patients with no RAVs), we will screen patients for RAVs in patients with HCV 
genotype 1a at the time of enrollment (20). Any patient with genotype 1a HCV found to 
have NS5A RAVs will undergo 16 weeks of therapy according to current treatment 
guidelines.  A complete panel of tests that will be performed during this visit is detailed in 
Appendix 1. 
 
Starting therapy 
Study drugs will be administered starting on day 0 after a history and physical examination 
is performed, eligibility is confirmed and clinical and research labs are collected.  A 
complete panel of tests that will be performed during this visit is detailed in Appendix 1. 
 
Study visits during treatment 
Patients will be followed every 4 to 8 weeks while they are receiving study drugs. Patients 
will be advised about study adherence and monitored for adverse events. A complete panel 
of tests that will be performed during this visit is detailed in Appendix 1.  Study medications 
will be distributed at visits. 
 
Safety and adverse event monitoring 
At each study visit, study staff will inquire about adverse events that may or may not be 
related to study drugs. Any unfavorable medical occurrences will be recorded, whether or 
not considered related to the patient’s participation in the research, temporally associated 
with the patient’s participation in the research. Adverse events (AEs) classified as grade 3 
or higher will be reported to the principal investigator.  Any grade 3 or 4 AEs and all SAEs 
will be reviewed as they occur by the study team (21).  Any AEs occurring more frequently 
than expected will be reported to the IRB. 
 
Principal Investigator shall forward to Merck’s Global Safety group, any SAE or Suspected 
Unexpected Serious Adverse Reaction (SUSAR), including, but not limited to, all initial and 
follow-up information involving any study subject in the study.  Notification shall be in the 

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form of a completed CIOMS I/MedWatch within two (2) business days of learning of the 
information. SAE reports and any other relevant safety information are to be forwarded to 
Merck’s Global Safety group facsimile number:  215-993-1220.  SAE and Reportable New 
Information will be reported to the IRB as per University policy. 
 
All reports of Study Drug exposure during pregnancy or lactation, whether associated with 
an AE or not, must be reported to Merck’s Global Safety group in accordance with the 
timelines and contact information for an SAE.  Principal Investigator shall follow 
pregnancies to term to obtain the outcome of the pregnancy.  The outcome of the 
pregnancy shall be forwarded to Merck’s Global Safety group. 
 
Clinical labs will also be drawn at these visits. There is a known, defined drug interaction 
between grazoprevir and tacrolimus causing an increase in tacrolimus levels of 
approximately 40%, as documented in the package insert (22).Levels of 
immunosuppressive agents will also be determined at these visits as clinically indicated.  
The need for dose modification of the patient’s immunosuppression in the time between 
visits will be recorded.  
 
End of treatment visit 
 Patients will be seen 12 weeks after starting study drugs (and 16 weeks in the case of 
genotype 1a patients with baseline NS5A RAVs) for an end of study visit. Patients will also 
be counseled about study adherence and we will inquire about adverse events. A complete 
panel of tests that will be performed during this visit is detailed in Appendix 1. 
 
Post treatment follow up visits 
Patients will be followed 12 weeks after they complete treatment. A complete panel of tests 
that will be performed during this visit is detailed in Appendix 1. 
 
Additionally, patients may be enrolled in an ongoing study titled “A prospective cohort study 
to assess treatment efficacy in chronic hepatitis C mono-infection and co-infection with HIV” 
(HP – 00063362), to monitor longer-term outcomes.  Outcomes including graft survival and 
episodes of rejection will be monitored by periodic medical record abstraction. 
 
Early termination or treatment discontinuation 
 Patients who discontinue HCV therapy prior to 12 weeks will be followed at post-treatment 
week 4 and post-treatment week 12. HCV VL, safety labs and hepatic panel will be 
performed at these visits. A complete panel of tests that will be performed during this visit is 
detailed in Appendix 1.  
 
2.5.2 Host Response Measurement  
 
Determination of Interferon stimulated gene expression associated with HCV 
suppression 
 
PCR.  ISG expression rapidly shuts down with initiation of DAA therapy in HCV infected 
subjects. We would like to evaluate the effectiveness of the host response to DAA therapy 
in pre- and post-transplant (immunosuppressed) patients by evaluating the changes in ISG 
expression observed in the two groups of patients before and 4 weeks after completion of 
HCV therapy.   
 
Expression analysis of select genes will be performed with predesigned or custom TaqMan 
assays individually or assembled into custom-designed 96-well plates or 384-well 
microfluidic cards (Life Technologies). Total RNA isolated from paxgenes will be reverse 
transcribed using random primers with the High Capacity cDNA Reverse Transcriptase Kit 
(Life Technologies). 1–25 ng RNA will be used for each qRT-PCR reaction. Gene 
expression will be determined as Ct based on 40 PCR cycles. For statistical analysis, 

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undetectable expression will be assigned a minimal detectable level with Ct of 40. 
Expression of GAPDH will be used as an endogenous control. Relative expression of 
targets normalized by GAPDH expression (ΔCt) will be calculated as CtGAPDH – Cttarget, with 
conversion and display relative to GAPDH expression by 2ΔCt. ΔΔCt values, used to 
calculate changes in expression between samples or groups of samples, will be calculated 
as ΔCtsample A – ΔCtsample B, then converted to a fold change by 2–ΔΔCt. Relative expression of 
individual ISGs FNs in blood will also be calculated as a percent of the sum of the total 
measured ISGs ([2ΔCt of each individual ISG/ total 2ΔCt of all measured ISGs] × 100). 384-
well microfluidic cards will be run on a 7900HT Fast Real-Time PCR System (Life 
Technologies). 
 
Cytokine and Chemokine ELISA.  IP-10 is the most studied soluble ISG that has been 
associated with response to therapy. The expression of IP-10 is rapidly shut down with 
initiation of DAA therapy in HCV infected subjects and a higher pre-treatment IP-10 level 
has been associated with a poorer response to therapy. We would like to evaluate the 
effectiveness of host response to DAA therapy in pre- and post-transplant 
(immunosuppressed patients) by evaluating the fold changes in IP-10 and other 
proinflammatory/chemotactic cytokine expression observed in the two groups of patients 
before and 4 weeks after completion of HCV therapy.  
 
Whole blood will be allowed to clot in for at least 60 minutes prior to centrifugation at 1,700 
g for 15 minutes. Serum will be collected and aliquoted prior to freezing at –80°C. After 
thawing, serum will be centrifuged at 1,450 g for 10 minutes, and the supernatant will be 
used for ELISA. Quantitation performed with multiplex (Chemokine 9-plex, Proinflammatory 
9-plex) assays that include CXCL10 (MesoScale Discovery). Plate-to-plate variability of 
pooled samples will be used for normalization across experiments. All samples will be run 
as technical duplicates, and averages will be used for data analysis. 
 
Determination of HCV specific CD4 and CD8 T cells by ELISPOT 
 
We have previously described an augmentation of HCV-specific T-cell responses with DAA 
therapy being associated with achieving an SVR 12. HCV specific T-cell phenotype and 
function will be characterized as previously described and measured before and at SVR 12.  
  
In brief, peripheral T-cells will be isolated from PBMCs by Ficollhypaque density gradient 
separation and be stimulated with pooled HCV genotype specific peptides.  For this 
experiment, we will use HCV 15-18-mer peptides with 11 or 12 amino acid overlaps 
spanning the entire HCV polyprotein and reconstituted in 5% sterile dimethylsulphoxide 
(DMSO), pooled consecutively into twenty-one groups and aliquoted until use. The number 
of HCV-responsive IFN-γ-producing PBMCs will be quantified by a standard ELISPOT 
assay (BD Biosciences), in which 96 well ELISPOT plates are coated with anti-IFN-γ 
biotinylated capture antibody and incubated overnight at 4°C. Plates will be blocked using a 
lymphocyte medium, and PBMCs will be allowed to rest for 6 hours at 37°C.  PBMCs will 
then be plated between 250,000- 400,000 cells per well with either phytohaemagglutinin 
(PHA) as a positive control (5 mg/ml), DMSO as a negative control (0.05%), or pooled HBV 
peptides (3 mg/ml/peptide). All cultures will be performed in duplicate. After incubating for 
12 hours at 37°C, cells will be removed, and plated with streptavidin detection antibody, 
enzyme conjugate, and substrate. The plates will then be air dried in the dark overnight, 
and developed spots will be enumerated using an ELISPOT plate reader. 
 
2.6 Study 
Duration 
We anticipate the entire study duration to be 24 months.  The duration per study subject 
enrolled will be 30 to 34 weeks depending on their treatment duration. This will include 6 
weeks of screening, 12 to 16 weeks of therapy, and 12 weeks of follow up to determine 
SVR 12 (Figure 1).   
 

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After the initial 30-34 weeks, patients will be enrolled in our ongoing HCV treatment cohort 
study (HP – 00063362).  Using this long term prospective cohort study, we will be able to 
follow our patients’ longer-term outcomes through medical record abstraction.  
 
2.7 Statistical 
Analysis and 
Sample Size 
Justification  
Data analysis will be completed by the study biostatistician. 
 
Primary Endpoints 
 
Primary Objective 1.  The primary endpoint for this objective will be the measurement of 
host immune responses using ISG expression including IP-10 levels, and HCV specific T-
cell responses. ISG expression and IP-10 levels will be measured before and 4 weeks after 
completion of therapy. HCV specific T-cell responses will be measured before and during 
SVR 12 (12 weeks after completion of the study medications). 
 
Primary Objective 2.  The primary endpoint for this objective will be the measurement of 
SVR 12.  This will be measured at study week 24/28, or 12 weeks after completion of the 
study medications. 
 
Secondary Endpoints 
 
Secondary Objective 1. We will assess the safety of the medications using adverse event 
monitoring.  The primary endpoint for this objective will be the presence of any severe 
adverse event.  A secondary endpoint will be the early discontinuation of the study 
medications.  This will be assessed at the study visits weeks 0, 4, 8, and 12.  Based upon 
prior trials with these investigational drugs, we estimate rate of serious adverse events of 
about 15% (17). 
 
Secondary Objective 2. The primary endpoint for this objective will be episodes of 
rejection and graft survival for post-transplant patients after treatment and will be recorded 
via medical record abstraction as part of their enrollment in the ongoing prospective cohort 
study to assess treatment efficacy in chronic hepatitis C (HP – 00063362). 
 
Research data will be collected and managed using REDCap (Research Electronic Data 
Capture) hosted at the University of Maryland, which is a secure, web-based application to 
support data capture for research studies. All of these variables will be entered into 
REDCap by study staff at the end of each study visit based upon the labs obtained, patient 
questionnaires, and interim records reviews, and after the initial 30 to 34 weeks of the study 
based upon medical records reviews. 
 
Statistical Methods 
 
Clinical treatment arm 
 
All statistical analyses will be based on an intention-to-treat model, including all enrolled 
patients who received at least one dose of study medication. Both the primary efficacy and 
safety endpoints will be based on the proportion of patients with SVR 12 and severe 
adverse events, respectively, with point estimates and 95% confidence interval. Both 
primary efficacy and safety endpoints will be compared to a non-transplant, HCV-infected 
historical control group by using two-sided, exact one-sample binomial test at a significance 
level of 0.05. Demographic and baseline characteristics will be summarized using standard 
descriptive statistics overall and by patient cohort.  Univariate logistic regression will be 
performed to identify baseline factors associated with SVR 12 at a significance level of 
0.05. 
 
Measurement of host immune response 

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Mann-Whitney or Wilcoxon matched-pairs signed rank test will be used for 2-group 
comparisons, and a linear mixed-effects model will be used for multigroup longitudinal 
comparisons with fixed effects for group (SVR versus relapse), time, and group/time 
interaction. A significant group/time interaction would indicate that the 2 groups had 
different patterns over time. A class variable will be used for time to allow an arbitrary 
average time pattern within a group. In addition to fixed effects summarizing average group 
responses over time, the model will accommodate random, patient-specific deviations in 
intercept and time effects. Correlations will be assessed by nonparametric Spearman rank 
correlation. Spotfire S+ 8.2 (TIBCO), Prism 6.0 software (GraphPad), and SAS 9.3 (SAS 
Institute, Inc.) will be used for statistical analysis and data presentation using a p-value of 
0.05 to indicate statistical significance. 
Power/Sample Size: 
This is a pilot, prospective clinical trial comparing the host immune response using ISG 
expression, including IP-10 levels, and HCV specific T-cell responses between a historical, 
non-transplant cohort of 25 patients from IRB approved studies previously conducted at the 
IHV and post-renal transplant (immunosuppressed) patients and its association with SVR 
12.  The study team will perform an interim data and safety evaluation for the efficacy, 
safety, and tolerability of grazoprevir and elbasivir therapy.  Data analysis will focus on the 
clinical efficacy endpoint of SVR 12 with secondary analysis looking at the relationship of 
the host immune response markers with the primary endpoint of SVR 12 in both groups. 
 
We anticipate enrollment of 25 patients in this arm of the study.  This will be compared to a 
historical control group of 25 patients with stored blood samples from previous IRB 
approved clinical trials conducted at the IHV. 
 
We anticipate a SVR 12 of greater than 90% in the grazoprevir plus elbasvir treatment 
group post-renal transplantation.  With 50 participants (25 in this study and 25 historical 
controls), the study will be able to estimate the difference in SVR12 proportions to within 
±0.18 (Table 2).  Table 2 shows a substantial gain in precision by increasing the sample 
size from 15 per arm to 25 per arm; however, the effect diminishes beyond 25. 
 
Table 2: Sample Size Calculation for  SVR12 
N per arm 
15 
20 
25 
30 
50 
Accuracy to within ± 
0.24 
0.20 
0.18 
0.17 
0.13 
2.8 Specific 
Drug Supply 
Requirements  
 
We request study drug supply from Merck Inc. for all study subjects. The study drugs will 
be received and distributed by Lisa Langer, RPh, through the UMD investigational drug 
pharmacy. The study drugs will be stored under the conditions specified by the 
manufacturer.  Packaging and labeling will be done by Merck.  At the study’s conclusion the 
remaining investigational product will be disposed of according to the ICH/GCP guidelines 
and our institution’s policies. 
 
2.9 Adverse 
Experience 
Reporting 
 
2.9.1 Overview 
 
Adverse experience reporting will be performed per study agreement at each study visit.  
During each clinical evaluation with the patients, information regarding adverse events and 
adverse reactions will be elicited.  Any untoward or unfavorable medical occurrences will be 
recorded, whether or not considered related to the patient’s participation in the research. 
Additionally, patients may be seen at unscheduled visits for a grade 3 or 4 adverse event or 
any unexpected adverse event or potential toxicity. 
 

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Adverse events (AEs) classified as serious or grade 3 or higher will be reported to the 
principal investigator.  Any grade 3 or 4 AEs and all SAEs will be reviewed as they occur by 
the study team.  Any AEs found to be occurring above the level expected by the study team 
will be reported to the IRB and study sponsor. 
 
For this study, patients may have expected AEs related to transplantation in the post-
transplantation arm or related to renal failure in the pre-transplantation arm.  This protocol 
will record all AEs possibly related to the treatment of HCV as defined by the Office for 
Human Research Protections (OHRP) (21). 
 
2.9.2 Definitions 
 
Adverse Event (AE): For this study, an adverse event will include any untoward or 
unfavorable medical occurrence associated with the patient’s participation in the research 
including, but not limited to: 
 
Worsening (a change in nature, severity or frequency) of medical conditions 
present at the onset of the study, including renal and liver dysfunction 
 
The development of new illnesses 
 
Any infections 
 
Abnormal laboratory values (significant shifts from the baseline values within the 
range of normal obtained at screening that the investigator considers clinically 
relevant) 
 
Clinically significant abnormalities in physical examination, vital signs, weight 
changes, and/or laboratory tests 
 
Subjective symptom reporting while on the study medication 
 
Serious Adverse Event (SAE): An adverse event or adverse reaction (AR) is considered 
“serious” if, in the view of the investigator it results in any of the following outcomes 
(adapted from FDA regulations at 21 CFR 312.32(a).) (21): 
 
Death 
 
A life-threatening event: an AE or AR is considered life-threatening if, in the view of 
the investigator, it places the patient at immediate risk of death.  This does not 
include AEs or ARs that, had it occurred in a more severe form, may have caused 
death. 
 
Inpatient hospitalization or prolongation of an existing hospitalization 
 
Persistent or significant disability or incapacity 
 
Congenital anomaly or birth defect 
 
Important medical events that may not fall into the above categories may be 
considered serious when, based upon appropriate clinical judgement, they may 
jeopardize the participant and may require medical or surgical intervention to 
prevent one of the above listed outcomes 
 
2.9.3 Grading and Attribution of Adverse Events 
 
2.9.3.1 Grading Criteria 
Adverse events will be graded according to the criteria set forth in the National Cancer 
Institute’s Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 (NCI-
CTCAE manual).  Adverse events will be graded on a scale from 1 to 5 according to the 
following standards (23): 
 
 
Grade 1 = mild adverse event 
 
Grade 2 = moderate adverse event 
 
Grade 3 = severe and undesirable adverse event 
 
Grade 4 = live-threatening or disabling adverse event 
 
Grade 5 = death 

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June 8, 2018 
 
2.9.3.2 Attribution Definitions 
The relationship or attribution of an adverse event to participation in the study will initially 
be determined by the investigator.  An AE will be defined as possible if the adverse event 
has a reasonable possibility of being related to study participation and/or there is evidence 
to suggest a causal relationship.  An AE will be defined as definite if the adverse event is 
clearly related to study participation.  An AE will be defined as unrelated if the adverse 
event is clearly not related to study participation and/or there is insufficient evidence to 
suggest a causal relationship (21). 
 
2.9.4 Collection and Recording of Adverse Events 
 
2.9.4.1 Collection Period 
Adverse events data grade 1 and higher will be collected from the time or initiation of study 
drug until a patient completes study participation or until 30 days after the patient withdraws 
from the study.  All Grade 3 or higher AEs will be reviewed by the investigator and reported 
to the sponsor and IRB if they are found to occur more frequently than expected by the 
study team and are related to study participation or the study drug. 
 
2.9.4.2 Collecting Adverse Events 
Adverse events may be identified during study visits through: 
 
 
Observation of the patient 
 
Interview 
 
Unsolicited complaints from the patient 
 
Abnormal clinical or laboratory values 
 
2.9.4.3 Recording Adverse Events 
At each study visit, the investigator will review all grade 1 or higher AEs and, if potentially 
related to study participation or the study drug, will report them to the study primary 
investigator.  An AE or SAE will be followed until resolution with or without sequelae, or 
until the end of study participation or 30 days after participant withdrawal, whichever comes 
first. 
 
2.9.5 Reporting of Serious Adverse Events and Adverse Events 
All SAEs determined to be possibly or definitely related to study participation or the study 
drug will be reported to the IRB and the study Sponsor (Merck Inc.).   
 
The IRB will be promptly notified of any unanticipated problems involving risks to 
participants or others, not otherwise reported as an adverse event. 
 
2.9.6  Data Safety Monitoring Board 
For this clinical trial, a Data Safety Monitoring Board (DSMB) led by an independent chair in 
collaboration with the PI and co-investigators will be utilized.  The DSMB will review this 
trial for safety in both the pre-transplant and post-transplant groups.  The first DSMB 
meeting will occur six months from the time of the enrollment of the first participant and 
then every six months thereafter.  Additionally, the board may be convened ad hoc if an 
adverse event calls for immediate attention. 
 
The DSMB will review the following information: 
 
Any adverse events 
 
Any safety related amendments 
 
Significant Protocol deviations 
 
Lab tests 
 
Enrollment 
 
Outcomes 

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 
Chart audit as needed 
 
The DSMB may require changes in the protocol and has the power to halt the study if a 
participant’s safety is ever in question.  Board members will be notified of any serious study 
related adverse events. 
 
The DSMB will be composed of at least four members: 
1. An independent physician-investigator not involved in the study who will serve as 
chair 
2. A physician-investigator with expertise in infectious diseases including hepatitis C 
treatment in renal transplant patients 
3. A physician-investigator with expertise in transplant nephrology in hepatitis C 
infected patients 
4. An epidemiologist with extensive training in biostatistics. 
 
2.10 Itemized 
Study Budget  
A refined itemized budget detailing the costs associated with the study was provided with the 
final protocol. 
2.11 
References 
 
1. Bunchorntavakul C, Maneerattanaport M, Chavalitdhamrong D. Management of patients 
with hepatitis C infection and renal disease.  World J Hepatol 2015;7:213-225. 
 
2.  Kirby BJ, Symonds WT, Kearney BP, and Mathias AA. Pharmacokinetic, 
Pharmacodynamic, and Drug-Interaction Profile of the Hepatitis C Virus NS5B Polymerase 
Inhibitor Sofosbuvir. Clin Pharmacokinet 2015; epub. 
 
3.  Harvoni [package insert]. Foster City, CA: Gilead Sciences, Inc.; 2015. 
 
4.  Saxena V, Koraishy FM, Sise M, et al. Safety and efficacy of sofosbuvir-containing 
regimens in hepatitis C infected patients with reduced renal function: real-world experience 
from HCV-TARGET [abstract LP08]. J Hepatology 2015;62:S267. 
 
5.  Viekira Pak [package insert]. North Chicago, IL: AbbVie Inc.; 2015. 
 
6.  Cohen D, et al. Ombitasvir/ABT-450/Ritonavir and Dasabuvir with or without ribavirin in 
treatment-naïve HCV genotype 1-infected adults with chronic kidney disease [abstract]. 50th 
Meeting of the International Liver Congress, April 22-26, 2015, Vienna, Austria. 
 
7.  Lawitz E, Gane E, Pearlman B, et al. Efficacy and safety of 12 weeks versus 18 weeks 
of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for 
hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and 
patients with previous null response with or without cirrhosis (C-WORTHY): a randomized, 
open-label phase 2 trial. Lancet 2015;385:1075-86. 
 
8. Yeh WW, Feng H, Dunnington KM, et al. No clinically meaningful pharmacokinetic 
interactions between HCV inhibitors grazoprevir/elbasvir with tacrolimus, mycophenolate 
mofetil, and prednisone, but cyclosporine increases grazoprevir/elbasvir exposures in 
healthy subjects [abstract]. AASLD 2015, Boston, MA. 
 
9. Rahermann B. Pathogenesis of chronic viral hepatitis: differential roles of T cells and NK 
cells. Nat Med 2013;19(7):859-868. 
 
10. Wieland S, et al. Simultaneous detection of hepatitis C virus interferon stimulated gene 
expression in infected human liver. Hepatology 2014;59(6):2121-2130. 

      Merck MISP 
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June 8, 2018 
 
11.  Meissner EG, Wu D, Osinusi A, et al. Endogenous intrahepatic IFNs and association 
with IFN-free HCV treatment outcome. J Clin Invest 2014;124:3352-63. 
 
14. Lagging M, Romero AI, Westin J et al. IP-10 predicts viral response and therapeutic 
outcome in difficult-to-treat patients with HCV genotype 1 infection. Heptology 
2006;44:1617-1625. 
 
13. Chang K, Thimme R, Melpolder JJ et al. Differential CD4+ and CD8+ T-cell 
responsiveness in hepatitis C virus infection. Hepatology 2001;33:267-276. 
 
14. Gerlach JT, Diepolder HM, Jung MC et al. Recurrence of hepatitis C virus after loss of 
virus-specific CD4+ T-cell response in acute hepatitis C. Gastroenterology 1999;117:933-
941. 
 
15.  Diago M, Castellano G, Garcia-Samaniego J, et al. Association of pretreatment serum 
interferon gamma inducible protein 10 levels with sustained virological response to 
peginterferon plus ribavirin therapy in genotype 1 infected patients with chronic hepatitis C. 
Gut 2006;55:374-9. 
 
16.  Larrubia JR, Moreno-Cubero E, Miguel J, and Sanz-de-Villalobos E. Hepatitis C virus-
specific cytotoxic T cell response restoration after treatment-induced hepatitis C virus 
control. World J Gastroenterol 2015;21:3480-91. 
 
17.  Roth D, Nelson D, Bruchfeld A, et al. Grazoprevir plus elbasvir in treatment-naïve and 
treatment-experienced patients with hepatitis C virus genotype 1 infection and chronic 
kidney disease (the C-SURFER study). Lancet 2015;386:1537-1545. 
 
18.  Carbone M, Cockwell P, and Neuberger J. Hepatitis C and kidney transplantation. Int J 
Nephrol 2011;2011:1-17. 
 
19.  Yeh W, Fraser IP, Reitmann C, et al. Pharmacokinetic interaction of HCV protease 
inhibitor MK-5172 and ritonavir in healthy subjects [abstract 52]. HEPDART 2013: Fronteirs 
in Drug Development for Viral Hepatitis, December 8-12, 2013, Big Island, Hawaii. 
 
20.  Jacobson IM, Asante-Appiah E, Wong P, et al. Prevalence and impact of baseline 
NS5A resistance-associated variants (RAVs) on the efficacy of elbasvir/grazoprevir 
(EBR/GZR) against GT1a infection – 16 weeks vs 12 weeks [abstract], November 13-17, 
2015, Boston, MA. 
 
21. OHRP “Guidance on Reviewing and Reporting Unanticipated Problems Involving Risks 
to Participants or Others and Adverse Events” (1/15/07) 
http://www.hhs.gov/ohrp/sites/default/files/ohrp/policy/advevntguid.pdf 
 
22.  Zepatier [package insert]. Whitehouse Station, NJ: Merck & Co, Inc.; 2016. 
 
23. National Cancer Institute’s Common Terminology Criteria for Adverse Events version 
4.0 (6/14/10) http://ctep.cancer.gov/reporting/ctc.html 
2.12 
Publication 
Plan 
We anticipate submitting the results of the study for publication as soon as the final 
analysis of the primary outcomes is performed, within 3-6 months after the completion of 
the last study participant.  We anticipate two publications, to be published in peer-reviewed 
journals.  We also anticipate two abstract presentations, one likely after study enrollment is 
complete and another after the completion of the study and analysis. 
 

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2.13 
Curriculum 
Vitae 
 
Dated CVs for the PI, Co-investigators, and study coordinator were provided with the 
application materials. 
2.13 Protocol 
Submission 
for 
Investigator-
Initiated 
Studies  
This protocol was submitted directly by the study PI.