Effect of Interferon Alpha 2b Intensification on HIV-1 Residual Viremia in Individuals Suppressed on Antiretroviral Therapy
Phase: PHASE1
Conditions: HIV Infection
Sponsor: National Cancer Institute (NCI)

 
 
Effect of Interferon Alpha 2b Intensification on HIV-1 Residual 
Viremia in Individuals Suppressed on Antiretroviral Therapy 
 
 
 
Short Title: Potential for Interferon Therapy in HIV-infected and ARV- suppressed 
Participants (PITHA) 
 
 
 
 
 
 
 
Principal Investigators: Frank Maldarelli, NCI 
Deborah McMahon, U of Pittsburgh 
 
 
 
 
 
 
 
 
 
Collaborating Investigators:  
 
Ann Wiegand 
 
Mary Kearney 
 
John Coffin 
 
John Mellors 
 
Research Contact: Jennifer Bell 
 
 
Version 5.0: March 31, 2015 
 
 

 
 
 
TABLE OF CONTENTS 
 
 
 
TABLE OF ABBREVIATIONS ........................................................................................................... 3 
Précis ................................................................................................................................................... 5 
1 Introduction ................................................................................................................................ 6 
2 Objectives .................................................................................................................................... 9 
2.1 
Primary Objective ............................................................................................................................ 9 
2.2 
Secondary Objectives ...................................................................................................................... 9 
3 Study Overview ........................................................................................................................ 10 
3.1 
Specific Aims ................................................................................................................................... 10 
3.2 
Study Design and Methods .......................................................................................................... 10 
3.2.1 
Study Population .................................................................................................................................................. 10 
3.2.2 
End Points ................................................................................................................................................................ 11 
3.2.3 
Sample Size .............................................................................................................................................................. 11 
3.2.4 
Safety Analyses ...................................................................................................................................................... 11 
4 Subject Selection...................................................................................................................... 11 
4.1 
Inclusion Criteria ........................................................................................................................... 11 
4.2 
Exclusion Criteria .......................................................................................................................... 13 
5 Schedule of Visits and Procedures...................................................................................... 13 
5.1 
Screening (Days -58 to -28) ........................................................................................................ 14 
5.2 
Pre-entry Visit 1 (Day-27 to Day -1) ......................................................................................... 14 
5.3 
Pre-Entry Visit 2: Leukapheresis Visits (Day -14 to Day -1, Day 28) ............................... 15 
5.4 
Entry (Day 0)................................................................................................................................... 15 
5.5 
Intensification Phase: Visit 1 (Day 7), Visit 2 (Day 14), Visit 3 (Day 21) ........................ 15 
5.6 
Early Post-Intensification Phase: Visit 4 (Day 28) ............................................................... 16 
5.7 
Late Post-Intensification Phase: (Visits 5 to 9) ..................................................................... 16 
5.8 
Long-term Follow-up: Week 16 (Visit 10) .............................................................................. 16 
5.9 
Weeks 24 and 36 (Visits 11 and 12) ......................................................................................... 17 
5.10 
Week 48 (Final Visit, Study Discontinuation Visit) ........................................................... 17 
5.11 
Window Periods for Study Visits ............................................................................................ 17 
6 Concomitant Medications ..................................................................................................... 17 
7 Study Drug Administration .................................................................................................. 18 
7.1 
Drugs and Dosing .......................................................................................................................... 18 
7.2 
Monitoring Parameters ............................................................................................................... 18 
7.3 
Dose Modifications/Toxicities ................................................................................................... 18 
8 Management of Toxicities to Study Drug .......................................................................... 18 
8.1 
Grade 4 Toxicities.......................................................................................................................... 18 
8.2 
Toxicity Management ................................................................................................................... 18 
8.3 
Criteria for Study Permanent Drug Discontinuation........................................................... 19 
9 Laboratory Studies.................................................................................................................. 19 

 
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9.1 
Serum Chemistry and Hematology Profiles ........................................................................... 19 
9.2 
Plasma HIV RNA Levels ................................................................................................................ 20 
9.3 
Pharmacokinetics of Pegylated Interferon ............................................................................ 21 
9.4 
Peripheral Blood Mononuclear Cells Storage ........................................................................ 20 
9.5 
CD4 Counts ...................................................................................................................................... 20 
10 Statistical Considerations ................................................................................................... 20 
11 Sample Size Estimation ....................................................................................................... 21 
12 Hazards/Discomforts/Risks .............................................................................................. 22 
12.1 
Study Drugs and Procedures ................................................................................................... 22 
12.1.1 
Pegylated Interferon Alpha 2b ................................................................................................................... 22 
12.1.2 
Granulocyte Colony Stimulating Factor ................................................................................................. 23 
12.1.3 
Potential for Previously Unrecognized Drug Interactions ........................................................... 24 
12.1.4 
Potential for Adverse Effects from Leukapheresis .......................................................................... 24 
12.1.5 
Risks of Phlebotomy ........................................................................................................................................ 24 
12.2 
Human Subject Protections ...................................................................................................... 24 
13 Study Recruitment ................................................................................................................ 24 
13.1 
Gender, Ethnicity, and Race Consideration ......................................................................... 25 
13.2 
Children and Pregnant Women ............................................................................................... 25 
14 Benefits/Compensation/Alternatives............................................................................. 25 
15 Evaluation of Safety .............................................................................................................. 26 
Recording/Documentation ................................................................................................................... 26 
15.1 
Protocol-specific Adverse Events ........................................................................................... 26 
15.1.1 
Definitions of Adverse Events..................................................................................................................... 26 
15.1.2 
Adverse Event Reporting .............................................................................................................................. 28 
Expedited Reporting to the NIAID IRB...................................................................................................................... 28 
15.1.2.2 Waiver of Reporting Anticipated Protocol Deviations, Expected non-UP AEs and 
Deaths ........................................................................................................................................................................................ 28 
15.1.2.4         Type and Duration of the Follow-up of Subjects after Adverse Events ........... 29 
16 Monitoring .............................................................................................................................. 29 
16.1 
Stored Samples and Future Research .................................................................................... 29 
16.2 
Data Management Plan.............................................................................................................. 29 
17 Anonymity and Confidentiality ......................................................................................... 30 
18 References .............................................................................................................................. 31 
 ............................................................................................................................................................. 35 
 Appendix 2: Toxicity Severity Scale ......................................................................................... 36 
 
TABLE OF ABBREVIATIONS 
AIDS 
Acquired immunodeficiency Syndrome 
ART 
Antiretroviral therapy 
BDS 
Beck Depression Inventory Score 
DHHS 
Department of Health and Human Services 
FDA 
Food and Drug Administration 

 
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G-CSF 
Granulocyte colony stimulating factor 
HCV 
Hepatitis C virus 
HIV-1 
Human immunodeficiency virus type 1 
HRPP 
Human Research Protection Program 
IRB 
Institutional Review Board 
INF 
Interferon 
NCI 
National Cancer Institute 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NNRTI 
Non-nucleoside reverse transcriptase inhibitor 
PBMC 
Peripheral blood mononuclear cells 
PEG 
Polyethylene glycol 
PEG-INTRON 
Pegylated interferon alpha 2b 
PI 
Principal investigator 
PrI 
Protease inhibitor 
SCA 
Single copy assay (viral RNA) 
 
 

 
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Précis  
As a result of combination antiretroviral therapy (ART), morbidity and mortality 
from acquired immunodeficiency syndrome has declined significantly in the past 15 
years, at least in developed countries. Human immunodeficiency virus type 1 (HIV-
1) infected individuals now live longer, but must undergo continuous therapy that 
has substantial consequences on quality of life.  
 
ART suppresses HIV-1 viremia below the limits of detection in current commercial 
assays (c. 50 copies/mL plasma)., but HIV viremia persists even after prolonged 
suppressive therapy. The origin of this residual viremia is yet not clear, but data 
suggest that production from long lived HIV infected cells may contribute to 
viremia.  Antiretrovirals are extremely active against replicating cells, and can thus 
successfully stop viral replication, but have no effect on long-lived viral reservoirs of 
cells already infected with HIV-1 at the time antiretroviral therapy is initiated. As a 
result, new strategies are necessary to reduce or eradicate long-lived reservoirs.  
Interferon alpha is a natural cytokine with antiviral activity. Prior to the introduction 
of antiretroviral therapy, several studies demonstrated modest effect of interferon 
alpha in HIV-1 viremia in active cycles of infection in infected individuals. Interferon 
alpha was also effective in vitro in decreasing virus production from cells chronically 
infected with HIV-1. With the introduction of potent antiretroviral therapy, 
interferon was not developed as a direct anti-HIV drug. Interferon alpha is relatively 
effective in therapy of hepatitis C virus (HCV) infection, and has been used in HIV-
1/HCV coinfected individuals. Kottilil and coworkers in the Laboratory of 
Immunoregulation NIAID have shown a decrease in HIV-1 RNA levels in HCV 
coinfected participants treated with pegylated interferon alpha and ribavirin. In 
stored samples from that study, we conducted a retrospective trial on samples from 
participants with HIV-1 RNA levels of <50 copies/mL, showing a further reduction in 
residual viremia using an ultrasensitive Single Copy Assay (SCA) developed in our 
laboratory. As such the effects of interferon on HIV viremia and cell associated HIV 
RNA are of growing interest.  
In this protocol we will conduct a prospective, non-randomized, single arm, pilot 
study to investigate the effect of pegylated interferon alpha 2b on HIV-1 RNA levels 
as an additional drug in participants undergoing suppressive antiretroviral therapy 
with viral RNA levels suppressed to less than 50 copies/mL plasma. As patients may 
have levels of HIV RNA that are lower than our limit of detection, we will also 
investigate levels of HIV nucleic acid species in cells as well We will determine 
whether interferon alpha therapy will reduce residual viremia or cell associated HIV 
RNA in participants on suppressive ART, which will expand our understanding of 
persistent low-level viremia and the pathogenesis of HIV in infected individuals. 
 
 

 
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1 
Introduction 
The human immunodeficiency virus type 1 (HIV-1) is responsible for the 
development of acquired immunodeficiency syndrome (AIDS) that results in 
progressive immune system deterioration and death. Therapy treating HIV 
(antiretroviral therapy or ART) by inhibiting viral replication and allowing immune 
restoration, has dramatically reduced AIDS-related mortality, but is not curative 
(Palella, Delaney et al. 1998, Egger, May et al. 2002). After introduction of ART, 
viral RNA levels in the blood decline significantly. The decay of HIV-1 replication in 
the presence of an effective ARV follows a biphasic pathway (Shen and Siliciano 
2008): a first rapid decay of the cells that produce >90% of plasma virus is 
followed by a second slower decline in viremia. Several studies have demonstrated 
that virus declines in plasma because new rounds of virus replication are 
interrupted by ART and because the cells producing virus die. As a result, the 
decline in viremia reflects the death rate of virus-infected cells. The first phase of 
decline in viremia involves mostly activated CD4+ T lymphocytes that have a very 
short life, surviving only about 1 day; the second phase involves mostly infected 
macrophages and partially activated CD4+ lymphocytes that have an half life of 
about 2 weeks.  
 
Decline in viremia is measured by commercially available assays that measure HIV-
1 RNA. The current limit of plasma HIV-1-RNA detection is 50 copies/mL, and this is 
the threshold that international guidelines use as the gold standard for therapy 
efficacy (Clumeck, Pozniak et al. 2008, DHHS 2008). Using ultrasensitive HIV-1 
RNA assays, with sensitivity of HIV-1-RNA down to 1 copy/mL, it is possible to 
detect persistent HIV-1 viremia even in participants undergoing successful 
treatment for prolonged periods with ARV (Di Mascio, Dornadula et al. 2003, 
Maldarelli, Palmer et al. 2007). In participants who have undergone an effective 
ARV regimen for 7 years, we showed an additional third and a fourth phase of viral 
decay in these participants.  
 
The set point of residual viremia is predicted by the pre-therapy HIV-1 RNA level; 
participants with pre-therapy HIV-1 RNA levels greater than 100,000 copies/mL had 
persistent detectable HIV-1 RNA levels over time (Palmer, Maldarelli et al. 2008). 
The origin of this residual viremia is uncertain. One possible source is an ongoing 
replication of viral strains spreading infection to uninfected cells (Zhang, 
Ramratnam et al. 1999, Havlir, Strain et al. 2003). Ongoing replication may occur if 
drug therapy does not completely inhibit virus replication, or if virus replication 
takes place in anatomic sanctuary sites in the body where antiretroviral drugs do 
not penetrate. Alternatively, HIV-1 may be produced from viral genomes integrated 
in cells from long-lived HIV-1 reservoirs (Palmer, Maldarelli et al. 2008, Shen and 
Siliciano 2008). Antiretroviral intensification studies revealed no decrease in HIV 
RNA levels after introduction of an additional potent antiretroviral(Dinoso, Kim et al. 
2009, McMahon, Jones et al. 2010, Besson, McMahon et al. 2012), suggesting 
ongoing replication was not the source of persistent HIV present during therapy. 
Increases in 2LTR circles in some patients suggest that low level replication events 
may occur in some patients (Buzon, Massanella et al. 2010, Hatano, Hayes et al. 
2011, Hatano, Strain et al. 2013). These data suggest that although replication 
events may occur, the predominant source of persistent viremia during therapy 

 
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is chronic viral production from long-lived cells, and strategies other than 
conventional antiretroviral therapy are needed to eradicate HIV-1 from infected 
individuals. In order to investigate the nature of the HIV-1 reservoir we plan to use 
the intensification strategy with novel antiretroviral therapy.  
 
One possible candidate in this strategy is the antiviral immune modulator interferon 
alpha (INF). Interferon alphas (interferon-alpha 2a and 2b) are a class of 
compounds known for their antiviral, immune-activating and immune-suppressing 
properties (Tilg 1997, Patel and McHutchison 2001, Tilg and Kaser 2004, Stetson 
and Medzhitov 2006). The addition of polyethylene glycol (PEG) to interferon 
increases its half-life in the body. PEG-Interferon alpha 2b, administered with 
ribavirin, is Food and Drug Administration (FDA)-approved for the treatment of 
chronic hepatitis C. Several clinical trials have shown its efficacy (especially in 
association with ribavirin) against HCV (Lindsay, Trepo et al. 2001, Manns, 
McHutchison et al. 2001). In addition, interferon alpha 2b is FDA-approved for the 
treatment of: hairy cell leukemia, malignant melanoma, follicular lymphoma, 
condylomata acuminata, AIDS- related Kaposi sarcoma, and chronic hepatitis B and 
C. In vitro studies conducted with HCV-infected cell-culture systems showed that 
INF exerts its activity by inducing a non-virus-specific antiviral state within the cell 
by activating a number of INF-responsive genes (Sen 2001, Bekisz, Schmeisser et 
al. 2004, Jiang, Guo et al. 2008). Exogenous administration of INF has a similar 
inducing effect, leading to a rapid reduction of HCV-RNA levels (Guo, Bichko et al. 
2001, Zhu, Zhao et al. 2003). Treatment with INF 2b or its pegylated form and 
ribavirin resulted in a successful clearance of hepatitis C in infected participants 
(Lindsay, Trepo et al. 2001, Manns, McHutchison et al. 2001), and the level of 
expression of genes related to INF response, immune cell signaling, defense 
response, and cell death has been associated with treatment outcome (Lempicki, 
Polis et al. 2006).  
 
With respect to HIV, INF has shown antiviral activity both in vitro (Ho, Hartshorn et 
al. 1985) and in vivo (Lane, Kovacs et al. 1988, Hatzakis, Gargalianos et al. 2001). 
The major activity of INF against HIV is thought to be the inhibition of the release 
of progeny virions from host-infected cells (Poli, Orenstein et al. 1989, Gottlinger, 
Dorfman et al. 1991). This anti- HIV-1 activity is opposed by the viral protein Vpu 
(Gottlinger, Dorfman et al. 1993) and recently the cell protein that mediates viral 
retention was identified as an INF-induced protein, Bst-317 or tetherin (Neil, Zang 
et al. 2008). In addition it is known that retroviral restriction factors such as 
APOBEC3G and TRIM5a are INF INF-regulated genes and their induction might also 
play a role in INF antiviral activity (Goff 2004). A number of in vitro studies 
suggested that the effects of interferon were modest (2-8 fold reductions in viral 
RNA) but interestingly, the combination of interferon and early antiretrovirals, such 
as AZT, was markedly synergistic (Hartshorn, Vogt et al. 1987), including in vitro 
models of cells chronically infected with HIV-1. These results are particularly 
intriguing because in cultures of chronically infected cells, the majority of the cells 
produce HIV-1 from integrated proviruses, and there is little spreading infection; as 
a result, AZT has minimal or no direct antiviral effect, and interferon may have 
been the most potent antiviral in these experiments (Poli, Orenstein et al. 1989). 
Experiments with HIV-1 production from chronically infected cells may represent a 

 
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useful model for participants suppressed on antiretroviral therapy as experiments 
suggest the majority of the detectable virus is produced from cells with integrated 
proviruses. Although interferon has been studied in active spreading infections, 
little data are available from participants who are already suppressed on ARV.  
 
In the pre-ART era, INF clinical trials showed a degree of efficacy in reducing viral 
replication, p24 production, and slowing progression to AIDS (de Wit, Schattenkerk 
et al. 1988, Lane, Davey et al. 1990, Berglund, Engman et al. 1991). After 
development of combination ARV, however, INF was not developed as an 
antiretroviral, due to lower antiviral efficacy, inconvenient route of administration, 
and adverse affects (Lane, Davey et al. 1990, Dusheiko 1997). Recently, several 
groups have shown a substantial HIV RNA reduction in participants treated with the 
pegylated form of interferon alpha (PEG-INF) as part of chronic hepatitis C therapy 
(Torriani, Ribeiro et al. 2003, Talal, Shata et al. 2004, Neumann, Polis et al. 2007). 
In addition, Asmuth and colleagues recently studied the effect of pegylated 
interferon alpha on HIV viral load in HBV/HCV seronegative HIV-1-infected 
participants who were not receiving antiretroviral therapy. HIV-1 viremia declined 
by a mean 0.77 log10 copies/mL over 12 weeks (Asmuth D 2009).  
The mechanism of interferon effect on HIV viremia is uncertain. There is a 
substantial literature regarding the effects of interferon in retroviruses in vitro, and 
in HIV in particular(Pitha 2011). Interferon may induce expression of a number of 
innate immune responses, which have direct effects on a number of distinct steps in 
virus replication. Induction of the cellular gene APOBEC 3G and 3F can result in 
hypermutation of the HIV genome during reverse transcription(Goila-Gaur and 
Strebel 2008). Induction of the gene tetherin results in retention of HIV on the 
surface of infected cells and decreases in production of extracellular HIV 
virions(Perez-Caballero, Zang et al. 2009). The effects of interferon may be 
manifest as a decrease in peripheral viremia, and a corresponding accumulation of  
HIV RNA intracellularly  
 
Much of the information available regarding the effects of interferon in HIV 
infection has been obtained in patients co-infected with HCV. We performed a 
retrospective analysis of HIV-1 RNA levels in plasma stored from HIV-1/HCV 
coinfected participants enrolled in NIH clinical trials for chronic hepatitis C 
treatment. We selected participants undergoing antiretroviral therapy with HIV-1 
viral RNA suppressed to <50 copies/mL, and used only samples that were obtained 
within 4 months of initiating therapy. Plasma HIV-1 RNA from 5/6 participants 
undergoing interferon/ribavirin therapy was decreased in 5/6 participants. In this 
retrospective pilot study, we could not distinguish whether the decrease in viremia 
resulted from therapy with interferon or ribavirin, or the combination. Prior clinical 
studies have indicated no evidence of anti-HIV-1 activity of ribavirin, suggesting 
that interferon may represent the active agent responsible for this result. Although 
the number of participants is small, these findings suggest that INF might decrease 
HIV-1 RNA levels in participants undergoing a successful ARV regimen.  
There are limited data on the in vivo effects of interferon alone in individuals 
undergoing antiretroviral therapy. Montaner et al., studied the effects of pegylated 
interferon on plasma HIV RNA in individuals undergoing antiretroviral therapy who 
underwent a structured ART interruption(Azzoni, Foulkes et al. 2013). Decreases in 
cell associated HIV DNA were detected in a subset of patients; no differences in 

 
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plasma HIV levels  were detected (using an assay with a 4-5 copy limit of detection 
,approximately 30 fold higher than the single copy assay. Differences in levels of 
cell associated DNA were detected, but no studies of intracellular HIV RNA were 
performed(Azzoni, Foulkes et al. 2013).Recently intracellular HIV RNA may be 
recognized by intracellular toll like receptors,which are themselves induced by 
interferon. As such the effects of interferon on cell associated HIV RNA are of 
growing interest. Recently we have developed digital droplet methods to quantify 
HIV RNA levels in infected cells, including levels of unspliced, spliced, and short 
(tat- ) transcripts, which may be synthesized in latently infected cells not producing 
full length HIV RNA.  In combination with single genome sequencing, these 
approaches will be useful in investigating the effects of interferon alpha in HIV 
infected individuals, including determining overall levels of cell associated RNA, 
numbers of cells producing HIV RNA, and phylogenetic analysis of HIV RNA 
sequences in plasma and cells. This advance will permit us to investigate the effects 
of interferon in patients with and without detectable levels of HIV RNA in plasma; 
patients with levels of HIV RNA below levels of single copy detection are still be 
detectable by measurements of HIV RNA . 
In the present protocol, we will investigate the effects of interferon alpha 2b 
in reducing low level HIV viremia. We plan to conduct a non-randomized, open-
label, single arm, pilot clinical trial of participants with viremia suppressed on 
antiretroviral therapy to investigate whether the addition of interferon alpha 2b 
therapy for 1 month (1.5 µg/kg interferon alpha 2b subcutaneously weekly for a 
total of 4 doses) results in changes in HIV-1 RNA levels in plasma or in peripheral 
blood lymphocytes. For the purposes of this pilot study, we will study 10 patients 
with suppressed viremia. Previously, we have obtained useful information regarding 
the effects of PI, NNRTI, and raltegravir intensification on HIV-1 viremia using this 
sample size. In the study planned here, we will perform gene expression studies to 
characterize the cellular response to interferon therapy in HIV infection. We will 
investigate potential correlations between expression of interferon-inducible genes 
and response. We will be particularly interested in the relationship between the 
interferon responsive gene, tetherin (Perez-Caballero, Zang et al. 2009), and the 
response to interferon. 
 
 
2 
Objectives 
2.1 Primary Objective 
This is a non-randomized, single arm pilot study of 10 participants to investigate 
the effect of interferon alpha on HIV-1 production in plasma and peripheral blood 
lymphocytes from HIV-infected individuals with HIV-1 RNA levels below the limit of 
detection in commercial assays. 
 
2.2 Secondary Objectives 
The secondary objectives of this study are the following: 
1. Investigate HIV-1 genetic variation in individuals undergoing interferon 
therapy.  

 
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2. Investigate the safety and tolerability of PEG INF 2b in HIV-1 infected 
individuals taking FDA-approved antiretrovirals in combinations 
recommended by Department of Health and Human Services (DHHS) 
guidelines. 
 
This study will allow us to elucidate the relationship between the viral decay in 
response to treatment intensification in HIV-infected individuals with residual 
viremia on antiretroviral therapy. This information will provide further clarifications 
on the origin of residual viremia and could provide clues to the eradication of HIV-1 
infection. 
 
This is a 48-week study to evaluate the effect of administering interferon alpha 2b 
to individuals in with viral RNA levels <50 c/ml undergoing antiretroviral therapy. 
Individuals entered into the study will viral RNA and PBMC sampling determinations 
prior to interferon intensification. During the second phase of the study, they will 
receive pegylated interferon alpha 2b (PEG-INTRON) at a dose of 1.5 µg/kg 
subcutaneously once weekly for 4 weeks starting at Study Entry, then at Visits 1, 2, 
and 3, for a total of 4 doses. During the interferon intensification phase, 
participants will continue to receive their standard of care HIV regimen. Following 
completion of the interferon dosing, participants will be monitored through 48 
weeks and will remain on their standard HIV regimen. During all phases, 
participants will be assessed for viremia using standard and single copy viral load 
assays. In addition, CD4 and CD8 counts as well as interferon levels will be 
measured.  
 
3 
Study Overview 
3.1 Specific Aims 
The goal of this study is to investigate the ability of pegylated interferon alpha 2b to 
reduce residual viremia or cell associated HIV RNA in HIV-infected individuals who 
are receiving antiretroviral therapy. Viral RNA load will be measured using the 
sensitive single RNA copy per milliliter assay. 
 
We will also delineate the safety and toxicity profiles of pegylated interferon alpha 
2b when co-administered with antiretroviral therapy in HIV-1 infected individuals. 
 
3.2 Study Design and Methods 
3.2.1 Study Population 
Ten evaluable HIV-1 chronically infected individuals with viremia suppressed to 
below the limit of detection, on antiretroviral therapy will be enrolled in this trial. 
Individuals with CD4 counts greater than or equal to 300 cells/µL at screening will 
be eligible to participate. We plan to conduct this study at the National Institute of 
Allergy and Infectious Diseases (NIAID)/CCMD Clinic at the National Institutes of 
Health (NIH) Clinical Center, and at the AIDS Clinical Trials Unit, Division of 
Infectious Diseases, University of Pittsburgh (active enrollment at Pittsburgh was 
completed in 2014).  

 
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3.2.2 End Points 
Unless unacceptable drug-related toxicities emerge (see Appendix 2), clinical 
contraindications unrelated to drug therapy develop that compromise the health or 
safety of study participants, or one of the specific criteria for withdrawal from the 
study is met, all participants will be treated for 4 weeks. Participants will be 
followed through Week 48. 
 
3.2.2.1 
Primary End Points 
We will compare viral RNA levels in blood and peripheral blood lymphocytes drawn 
before, during, and after intensification, including comparison of absolute values of 
viremia, as well as changes in viremia and area under the curve analyses to detect 
whether increases or decreases of viremia occurred as a function of interferon 
therapy. Standard analyses will be included. 
 
3.2.2.2 
Secondary End Points 
In participants with detectable and amplifiable virus, HIV-1 genetic sequencing will 
be performed to determine whether interferon therapy results in differences in 
population structure during the 4-week intensification period. HIV RNA and DNA 
levels will be investigated using quantitative PCR approaches, including digital 
droplet PCR. The safety and tolerability of single agent interferon will be assessed 
through standard clinical management. 
 
3.2.3 Sample Size  
As described below (see Statistical Considerations) we expect that 10 participants 
completing the study will be necessary to yield useful clinical information. In order 
to enroll 10 participants for this study, we will be screening up to 65 individuals. 
Any dropouts and non-adherent participants will be replaced. 
 
3.2.4 Safety Analyses 
All safety data including adverse events, deaths, vital signs, physical examinations, 
discontinuations from study drug, and clinical laboratory measurements 
(hematology, serum chemistry, and urinalysis) will be listed and summarized, using 
the mean, standard deviations, and percentiles for continuous variables and 
frequencies and percentages for categorical variables. Any notable values will be 
flagged. Any comparisons with respect to safety parameters from baseline will be 
made using the same procedures described above for endpoint analyses. 
 
4 
Subject Selection 
4.1 Inclusion Criteria 
To be eligible for study participation, a volunteer must satisfy all of the following 
inclusion criteria: 

 
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1. Age >18 years. 
2. Documentation of HIV-1 infection by any licensed ELISA test and confirmed 
by a Western Blot. 
3. Receiving a DHHS-approved ARV regimen,. 
4. Level of cell-associated HIV RNA ≥ 5 copies/million peripheral blood 
mononuclear cells (PBMC) done at screening visit 1 
5. HIV-1 RNA levels less than detectable by current commercial means (e.g., 
Roche Amplicor, b-DNA test) for a minimum of 12 months prior to screening 
at all time points, and with at least 2 measurements in this 12 month 
window. 
 
6. CD4 >300 cells/mm3 at pre-entry visit within 14 days prior to enrollment. 
7. Ability to sign informed consent and willingness to comply with the study 
requirements and clinic policies. 
8. No evidence of viral hepatitis co-infection as assessed by Hepatitis C 
antibody, HCV RNA, and hepatitis B surface antigen; determinations at pre-
entry visit within 28 days prior to enrollment. 
9. No history of or evidence of autoimmune hepatitis or other autoimmune 
disorders at screening, or antinuclear antibody (ANA > 3x upper limit of 
normal. 
10. Laboratory values at pre-entry visit within 14 days prior to enrollment:  
• 
Alkaline phosphatase <2.0 times upper limit of normal 
• 
Alanine transaminase (ALT) <2.0 times upper limit of normal 
• 
Total bilirubin <2.5 mg/dL (< 40 mg/dL if on Atazanavir) 
• 
Creatinine clearance >60 mL/min as estimated by the Cockcroft-Gault 
equation 
• 
Neutrophil count >1500 cells/mm3 
• 
Platelets >150,000/ mm3 
• 
Hemoglobin >12.0 mg/dL for men and >11.0 g/dL for women 
• 
Fasting glucose <126 mg/dL 
11. No history or evidence of significant clinical depression at screening that in 
the opinion of the investigator would affect the ability of the patient to 
participate in the study, or which would constitute a threat for his/her health 
in case of relapse upon INF treatment. The Beck Depression Inventory score 
must be £13 at pre-entry visit. 
12. No history of INF/PEG-INF therapy. 
13. If capable of pregnancy: use of effective contraception during study: 
effective contraception methods include abstinence, surgical sterilization of 
either partner, barrier methods such as diaphragm, condom, cap or sponge, 
or use of hormonal contraception with an anti-HIV regimen that will not alter 
metabolism of hormonal contraception. 
14. Participants must have primary medical care outside this protocol: 
participants will have to provide Primary Care Doctor’s contact information. 
 

 
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4.2 Exclusion Criteria 
A volunteer will be ineligible to participate in this study if any of the following 
criteria are met: 
 
1. History of neoplastic disease requiring cytotoxic therapy including 
hydroxyurea. 
2. Use of long-term systemic corticosteroids, immunosuppressive agents, or 
cytotoxic agents within 60 days prior to enrollment. 
3. Any vaccination within 30 days prior to enrollment. Individuals interested in 
participating who require vaccination will delay screening until 30-day period 
is completed. 
4. Concurrent therapy with investigational cytokines including IL-2 or IL-12 
during the course of the study. Prior administration of cytokines is not an 
exclusion criterion; at least 4 months from most recent cycle of IL-2 or IL-12 
is required. 
5. Any febrile illness (>38oC) in the 3 weeks prior to enrollment or any acute 
therapy for a serious infection completed within 30 days prior to enrollment. 
6. Current pregnancy or lactation, history of pregnancy in the last 4 months. 
7. Preexisting autoimmune disorders including inflammatory bowel diseases, 
psoriasis, idiopathic thrombocytopenic purpura, lupus erythematous, 
autoimmune hemolytic anemia, scleroderma, severe psoriasis, rheumatoid 
arthritis, and optic neuritis. 
8. History of severe retinopathy or evidence of severe retinopathy judged by 
pre-entry ophthalmologic examination. 
9. Known allergy/sensitivity to study drug or its formulation. 
10. History of seizure disorders or current anticonvulsant use. 
11. Any history of medical conditions associated with chronic liver disease 
(genetic hemochromatosis, alcoholic liver disease, toxin exposures, and 
autoimmune hepatitis) or documented cirrhosis due to any cause. 
12. History of pulmonary disease associated with functional limitation. 
13. Documented history of thyroid disease. 
14. Active drug or alcohol use or dependence, which in the opinion of the 
investigator, would interfere with complying with the study requirements. 
15. Known hypersensitivity to Escherichia coli-derived products such as 
filgrastim. 
16. Any systemic illness that will make it unlikely that the subject will be able to 
return for the required study visits. 
17. History of, or any condition that in the opinion of the investigator would 
interfere with the conduct of the study, or it would not be in the best interest 
of the subject to enroll in this study. 
 
5 
Schedule of Visits and Procedures 
Participants will be seen in the NIAID/CCM Clinic at the NIH Clinical Center, NIH (PI: 
Frank Maldarelli, MD) OR at the AIDS Clinical Trials Unit, Division of Infectious 
Diseases, University of Pittsburgh (PI: Deborah McMahon, MD; active enrollment at 
Pittsburgh was completed in 2014). We plan to enroll a total of 10 completed 

 
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participants for this study.  Any dropouts and non-adherent participants will be 
replaced. 
 
5.1 Screening (Days -58 to -28) 
Screening will consist of HIV-1 RNA level and if < 50 copies/mL by standard assay 
and have detectable PBMC-associated HIV RNA  ≥ 5 copies/million cells will 
continue with screening. If > 50 copies/mL, the subject will not undergo further 
screening assessments.  
 
Screening for the protocol will be completed within 4 weeks prior to the study 
enrollment. During screening visit the protocol design will be reviewed, and 
informed consent will be obtained prior to any study-related screening procedures. 
The subject will then be given the consent document to read. Consent forms may 
be sent to the subject for review prior to the screening visit. The site investigator 
will review the study with the subject and answer any questions the subject may 
have about the study design, interferon, or study procedures. After the subject has 
read this information, there will be further opportunity for discussion with one of 
the investigators.  Potential participants will sign consent prior to any study-related 
procedures. Once consented, eligibility will be assessed.  
 
 
5.2 Pre-entry Visit 1 (Day-27 to Day -1)    
When eligibility evaluation is complete, the subject will return for further 
assessments and the standard consent will be administered. The subject’s medical 
history will be reviewed and a physical examination will be performed, including 
weight and vital signs. Concomitant medications will be documented. The 
assessments are summarized in Appendix 1, and include:  
• 
Complete physical examination, including vital signs 
• 
Beck Depression Inventory Scoring (BDS) 
• 
Complete blood count with differential, platelet count 
• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase) 
• 
Creatinine clearance (Cockcroft-Gault method) 
• 
Fasting blood glucose 
• 
TSH, free T4 
• 
Urine pregnancy test (women of childbearing age) 
• 
Antinuclear antibody (ANA) 
• 
Urinalysis 
• 
Amylase 
• 
ALT, bilirubin 
• 
Hepatitis B surface antigen, hepatitis B surface antibody, hepatitis C antibody 
and hepatitis C viral RNA level (HCV PCR) 
• 
CD4 count 
• 
Ophthalmology examination (dilated funduscopic examination) 
• 
Single copy assay for HIV-1 RNA 
• 
HIV-1 RNA standard assay 

 
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• 
HIV-1 ELISA/Western Blot (if documentation not available) 
 
5.3 Pre-Entry Visit 2: Leukapheresis Visits (Day -14 to Day -1, Day 28) 
Once the subject has been found to meet the eligibility criteria, the subject will 
return for leukapheresis for the purposes of peripheral blood lymphocyte (PBL) 
storage for use in future virologic and immunologic studies. Leukapheresis, will be 
scheduled to occur at Pre-Entry 2 and during Visit 4 (Day 28). 
 
5.4 Entry (Day 0) 
Participants will have laboratory safety assessments performed and receive their 
first dose of PEG-IFN. Administration of pegylated interferon will be performed by 
trained personnel. Vital signs will be obtained, concomitant medications 
documented, and a targeted physical examination will be performed based upon 
any signs or symptoms that the subject has experienced since the last visit. 
Assessments are detailed in Appendix 1 and include: 
• 
Beck Depression Inventory score 
• 
Complete blood count with differential, platelet count 
• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase) 
• 
Urine pregnancy test (women of childbearing age) prior to interferon 
administration 
• 
ALT, Bilirubin 
• 
Creatinine clearance (Cockcroft-Gault method) 
• 
CD4 count 
• 
Single copy assay for HIV-1 RNA 
• 
HIV-1 RNA standard assay 
• 
Interferon level (baseline) 
• 
PBMCs for storage 
 
5.5 Intensification Phase: Visit 1 (Day 7), Visit 2 (Day 14), Visit 3 (Day 
21) 
Participants will have laboratory safety assessments performed and receive their 
subsequent doses of PEG-IFN. Vital signs will be obtained and concomitant 
medications documented. Administration of interferon will be performed by study 
personnel. Dosing will be based on body weight obtained during initial evaluation. 
The last PEG-IFN administration will be on Day 21 (Visit 3). A targeted physical 
examination will be performed based upon any signs or symptoms that the subject 
has experienced since the last visit. Assessments are detailed in Appendix 1 and 
include: 
• 
Beck Depression Inventory score 
• 
Complete blood count with differential, platelet count (STAT, prior to 
interferon administration) 
• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase) 
• 
Creatinine clearance (Cockcroft-Gault method) 

 
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• 
Urine pregnancy test (women of childbearing age) prior to drug 
administration 
• 
ALT, Bilirubin 
• 
Single copy assay of HIV-1 RNA 
• 
HIV-1 RNA standard assay 
• 
Interferon level 
 
5.6 Early Post-Intensification Phase: Visit 4 (Day 28) 
Participants will have laboratory safety assessments performed. Vital signs will be 
obtained and concomitant medications documented. A targeted physical 
examination will be performed based upon any signs or symptoms that the subject 
has experienced since the last visit. Assessments are detailed in Appendix 1 and 
include: 
• 
Beck Depression Inventory score 
• 
Complete blood count with differential, platelet count  
• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase) 
• 
Creatinine clearance (Cockcroft-Gault method) 
• 
ALT, Bilirubin 
• 
CD4 count 
• 
Single copy assay of HIV-1 RNA 
• 
HIV-1 RNA standard assay 
• 
Interferon level 
• 
PBMCs for storage 
• 
Leukapheresis 
 
5.7 Late Post-Intensification Phase: (Visits 5 to 9) 
Participants will return for visits at Days 35, 42, 49, and 56 during the pegylated 
interferon post-intensification phase. The Assessments are detailed in Appendix 1 
and include: 
• 
Beck Depression Inventory score 
• 
Complete blood count with differential, platelet count  
• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase) 
• 
Creatinine clearance (Cockcroft-Gault method) 
• 
ALT, bilirubin  
• 
Single copy assay for HIV-1 RNA 
• 
HIV-1 RNA standard assay 
• 
CD4 count (Visits 8 and 9) 
 
5.8 Long-term Follow-up: Week 16 (Visit 10) 
The following determinations will be assessed at the Week 16 visit: 
• 
Beck Depression Inventory score 
• 
Complete blood count with differential, platelet count  

 
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• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase)  
• 
Creatinine clearance (Cockcroft-Gault method) 
• 
ALT, bilirubin  
• 
Single copy assay for HIV-1 RNA and assays for cell associated HIV 
• 
HIV-1 RNA standard assay 
 
5.9 Weeks 24 and 36 (Visits 11 and 12) 
The following determinations will be assessed at the Week 24 and Week 36 visits: 
• 
HIV-1 RNA standard assay 
• 
Single copy assay for HIV-1 RNA and assays for cell associated HIV 
 
5.10 Week 48 (Final Visit, Study Discontinuation Visit) 
The following determinations will be assessed at the Week 48 or Study 
Discontinuation visit: 
• 
Beck Depression Inventory score 
• 
Complete blood count with differential, platelet count  
• 
Serum chemistries (sodium, potassium, chloride, carbon dioxide, creatinine, 
urea nitrogen, alkaline phosphatase)  
• 
Creatinine clearance (Cockcroft-Gault method) 
• 
ALT, bilirubin  
• 
Single copy assay for HIV-1 RNA and assays for cell associated HIV 
• 
HIV-1 RNA standard assay 
• 
CD4 count 
• 
PBMC 
 
5.11 Window Periods for Study Visits 
The following variable scheduling is permitted for study visits: 
Days -58 to 14: ±1 day  
Days 21 to 49: ±2 days  
Days 56 to 84: ±5 days  
Weeks 16 to 48: ±14 days  
 
6 
Concomitant Medications 
All concomitant medications will be documented at each study visit. 
 
Concomitant administration of immunomodulatory treatments (including steroids, 
radiation, IL-2, and anti-neoplastic agents) is not allowed during the course of this 
trial. Other investigational drugs are excluded. No vaccinations are allowed during 
the study period from 30 days  prior to the time of signing consent to the end of  
the four week interferon therapy period.  
.  
 

 
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7 
Study Drug Administration 
7.1 Drugs and Dosing 
All participants will receive pegylated interferon alpha 2b at the dose of 1.5 µg/kg 
subcutaneously every week for 4 doses. Dosing will be determined at Entry, and 
Visits 1, 2, and 3 based on subject's body weight obtained at the time of 
enrollment. Administration of study medication will be performed by study 
personnel. Antiretroviral therapy will be continued throughout the study. 
 
7.2 Monitoring Parameters 
The Schedule of Events is found in Appendix 1. Participants will be closely 
monitored for clinical and laboratory evidence of adverse events associated with the 
study drug or medical complications. 
 
7.3 Dose Modifications/Toxicities 
A complete blood count with differential (done STAT) will be reviewed before PEG-
INF administration at visits 0, 1, 2, and 3. Participants experiencing an adverse 
event Grade £2 may continue at their current dose. No interferon dose 
modifications will be made in the study but additional visits or monitoring will be 
permitted to evaluate participants. Toxicity will be evaluated using the Division of 
AIDS Table for Grading Adults Adverse Events:  
(http://rcc.tech-res.com/safetyandpharmacovigilance/ ).  
 
8 
Management of Toxicities to Study Drug 
8.1 Grade 4 Toxicities 
Participants with life threatening or Grade 4 adverse events thought to be possibly, 
probably, or definitely related to study treatment will discontinue study treatment 
and remain on study follow-up and undergo additional visits and therapy as needed 
for toxicity management. Grade 4 neutropenia determinations will be repeated with 
duplicate samples to document that neutropenia is present. 
 
8.2 Toxicity Management  
Specific expected toxicities lower than grade 4 will be managed as follows: 
 
Adverse Event Action 
Neutropenia 
Participants who experience Grade 3 neutropenia will receive 
filgrastim at 300 µg subcutaneously up to 3 times a week. Absolute 
neutrophil counts will be monitored until return to subject’s 
baseline or <Grade 1. Patient will be asked to return in 1-3 days for 
evaluation. If grade 3 neutropenia has resolved, patient will return 
2-3 days later for repeat evaluation. If grade 3 neutropenia has not 
resolved at the day 1-3 visit, additional G-CSF will be administered 

 
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and the patient will return daily for evaluation and additional G-CSF 
as medically indicated until resolved.  Filgrastim will be 
discontinued at this time. Interferon will be continued for Grade 3 
neutropenia. 
CD4 cell count If the CD4 count declines to <14%, pneumocystis prophylaxis will 
be offered until the percentage of CD4 cells increases to ≥14%.  
Depression 
If a BDS score of 14-19 is reported, assessment by the site 
investigator will be scheduled before the next dose of study drug. If 
a BDS score of 20-28 is reported, a psychiatric evaluation is 
required before the next dose of study drug. If a BDS score is >29 
or suicidal ideation is reported, participants will permanently 
discontinue study drug and undergo prompt psychiatric evaluation.  
 
 
8.3 
Criteria for Study Permanent Drug Discontinuation 
In addition to the criteria for stopping the study drug, the following will constitute 
criteria for discontinuation of study drug: 
 
1. Development of life-threatening infection or malignancy. 
2. Grade 3 or higher neutropenia. 
3. Development of clinical depression (see Section 8.5). 
4. Acquisition of hepatitis A, B, or C. 
5. Pregnancy. 
6. If subject’s CD4 cell percent remains <14% (see Section 8.2). 
7. If it is felt by the site’s principal investigator that further participation in the 
study is no longer in the best interest of the subject. 
8. Development of a medical condition, that in the opinion of the investigator, it 
is in the subject’s best interest to discontinue study treatment even if criteria 
requiring drug discontinuation have not been met. 
9. In addition, participants may be withdrawn from the study for: 
a. Withdrawal of consent 
b. Non-compliance with study visits or study treatment; subject misses a 
dose of study drug for reasons other than toxicity 
c. Termination by sponsor 
 
9 
Laboratory Studies 
9.1 Serum Chemistry and Hematology Profiles 
Routine safety laboratory tests (Appendix 1) will be performed to monitor clinical 
status and detect potential drug-related toxicities. Safety tests will be performed at 
each site’s laboratory. During the intensification phase of the study, results of 
safety tests will be reviewed by the site investigator at each visit before PEG-
Interferon administration. 
 

 
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9.2 HIV RNA Levels 
HIV-1 RNA levels will be measured by FDA-approved standard assay (b-DNA or HIV 
RT-PCR assay) and SCA assay will be measured at screening and at each study 
visit. Samples for b-DNA testing and samples for SCA assay will be processed the 
same day and plasma will be stored at -80°C. Analysis of HIV in plasma and cells 
will be analyzed through the Clinical Retrovirology Section of the HIV Drug 
Resistance Program and the HIV Molecular Monitoring Core (Leidos).  
 
9.3 Pharmacokinetics of Pegylated Interferon 
Serum levels of pegylated interferon alpha 2b will be measured at Entry and Visits 
1, 2, 3, and 4. 
 
9.4 Peripheral Blood Mononuclear Cells Storage 
PBMCs will be collected and stored at Entry, at Visits 4, , and at the Final Visit (13), 
or in the event of early study discontinuation. Samples will be processed according 
to standard processing for cryopreservation. 
 
9.5 CD4 Counts 
CD4 counts (absolute number and percentage) will be measured at Pre-entry, 
Entry, Visits 1, 4, 8, 9 and the Final Study or Discontinuation Visit. Samples will be 
processed according to standard flow cytometry procedures.  
 
10 Statistical Considerations 
 
 
This pilot study will investigate whether pegylated interferon alpha2-b will reduce 
residual HIV-1 RNA in participants with HIV-1 viremia suppressed below detectable 
levels  as measured by commercial assays.  Two relevant questions are: 1) does 
intensification reduce viremia in a consistent way across different patients, and 2) 
are there any patients in whom intensification reduces viremia?  The first question 
is of primary interest, and it pertains to the population.  The second question 
concerns whether individual intensification responders can be identified.  It is 
possible to find no consistent pattern across patients, yet identify one or more 
patients in whom the pattern of viremia lowering over time convincingly rules out a 
flat slope for that patient.  Therefore, analyses will be directed at both population 
responses and individual responses, with the primary emphasis being on the 
former. 
 
For both population and individual analyses, one complication is that because 
patients are selected on the basis of low baseline viral loads, regression to the 
mean would cause subsequent viral loads to be somewhat larger, in the absence of 
intensification.  To account for this, we will use one baseline measurement to 
determine eligibility and another to assess changes over time.   
 

 
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The primary analysis (which is at the population level), compares the viral loads 
obtained from the pre-intensification control period to the week 4 value using a 
paired t-test.  A sensitivity analysis will use the nonparametric Wicoxon signed rank 
test to analyze these baseline to week 4 changes.  Another analysis computes the 
slope of viral load over time for each patient, and uses a 1-sample t-statistic and 
confidence interval to estimate and test whether the average slope is 0.   
 
Secondary analyses will attempt to identify individual intensification responders. 
One definition of responder is whether the patient had at least a 1-log decrease 
from baseline to 4 weeks in viral load.  Because patients with baseline viral load 
below 10 cannot achieve this, another method will compute the least squares slope 
of viral load versus time for each patient and declare patients with statistically 
significant slopes as responders.   Because the goal for this analysis is to 
summarize the response of a given individual, we treat the data as independent 
observations from that patient.  This is the same assumption made when using a 
mixed model, namely that conditioned on the random, patient-specific intercept and 
slope, the observations are independent.  For both the 1 log method and significant 
slope method of identifying individual responders, we attempt to find differences in 
baseline characteristics between responders and non-responders, though very low 
power precludes finding any of these statistically significant unless they are huge.  
Of particular interest is whether baseline viral load predicts responder status.  As 
described above, for this analysis we will use one baseline value to characterize 
baseline viral load and another for the calculation of slope to identify responder 
status.  
 
All tests will be two-sided and an alpha level of less than 0.05 will be considered 
statistically significant.  Analyses will be performed on both intent-to-treat and as 
per protocol participants. Any dropouts and non-adherent participants will be 
replaced. We do not feel that stopping criteria are necessary as this pilot study is 
not for prognosis or clinical management; there is no blinding of study treatment 
and we will be monitoring toxicities in real time. In this study, we will be studying 
participants on an ongoing basis, and no interim analysis is planned. 
 
 
11 Sample Size Estimation  
This is a pilot study of the effect of pegylated interferon on HIV-1 RNA levels in 
participants who are receiving antiretroviral therapy. To date, there is limited 
published data available, and the proportion of participants who will respond with 
decreases in HIV-1 viral RNA levels remains to be clearly defined. As described 
above, responses may be similar to those observed with PI, integrase inhibitor, or 
NNRTI intensification, where no participants have experienced a decrease in viral 
RNA levels. If so, previous sample size analyses estimating the frequency of 
suppressible participants are relevant. In the accompanying table, we estimate the 
90% confidence intervals of the probability that a participant has a detectable 
decrease in viral RNA levels (participants suppressed in the table below) after 
studying increasing numbers of participants without observing episodes of drug 
suppression. As shown here, if we study 10 participants and observe no 

 
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suppression in plasma or cell-associated HIV RNA, we have 90% confidence that 
the frequency of suppressible participants is less than 21%. For this pilot study, we 
will request a sample size of 65 participants to achieve our goal of studying 10 
participants who have ≥0.1 copies/mL HIV-1 RNA at screening.  
 
90% exact confidence intervals (using the Clopper-Pearson method) for the 
probability of being suppressible.  
  
Participants 
intensified 
Participants 
suppressed 
 90% confidence interval 
5 
0 
0,0.45 
6 
0 
0,0.39 
7 
0 
0.0.35 
8 
0 
0,0.31 
9 
0 
0,0.28 
10 
0 
0,0.26 
15 
0 
0,0.18 
20 
0 
0,0.14 
 
 
 
12 Hazards/Discomforts/Risks 
12.1 Study Drugs and Procedures 
12.1.1 
Pegylated Interferon Alpha 2b 
Interferon alpha is a cytokine with proven antiviral activity against many viruses, 
especially HCV. The exact mechanism of this antiviral activity is not known. The 
proposed mechanisms of actions for interferon alpha are:  
1. Antiviral activity through the inhibition of viral replication in infected cells 
(Sen 2001, Bekisz, Schmeisser et al. 2004, Jiang, Guo et al. 2008). With 
respect to HIV-1 specifically, INF has shown antiviral activity both in vitro 
(Ho, Hartshorn et al. 1985) and in vivo (Lane, Kovacs et al. 1988, Hatzakis, 
Gargalianos et al. 2001) studies.  
2. Immune regulatory actions increasing the ability of reticuloendothelial cells to 
clear virus through the following mechanisms (Missale, Bertoni et al. 1996, 
Gerlach, Diepolder et al. 1999, Patel and McHutchison 2001):  
a. Increase in class 1 MHC antigens and cellular adhesion molecules on 
effector cells 
b. Activation of effector cells (NK cell and macrophages) 
c. Upregulation of Th1-T helper subset  
 
In participants with cirrhosis, exacerbation of cirrhosis-related neutropenia and 
thrombocytopenia is a recognized risk associated with interferon and pegylated 
interferon. Participants with documented cirrhosis will be excluded from 

 
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participating in this study. The other recognized side effects associated with the use 
of pegylated interferon include fatigue, headaches, myalgia, fever, nausea, upper 
abdominal discomfort, depression, diarrhea, insomnia, reduced appetite, and 
dermatitis. There are also rare occurrences of autoimmune, infections, ophthalmic, 
and ischemic disorders associated with pegylated interferon use. Ophthalmologic 
complications include retinopathy, which is usually transient and completely 
resolves with continued treatment. Other side effects associated with subcutaneous 
injection include pain and inflammation at the site of injection.  
The specific adverse events listed below will be handled as specified. PEGINTRON 
will be administered in clinic by trained personnel.  Merck Inc. will be providing 
study drug PEGINTRON; other study agents will be provided through respective 
pharmacies as indicated. 
12.1.2 
Granulocyte Colony Stimulating Factor 
Endogenous granulocyte colony stimulating factor (G-CSF) is a lineage specific 
colony-stimulating factor, which is produced by monocytes, fibroblasts, and 
endothelial cells. G-CSF regulates the production of neutrophils within the bone 
marrow and affects neutrophil progenitor proliferation, differentiation, and selected 
end-cell functional activation. 
Filgrastim is a human granulocyte colony-stimulating factor (G-CSF), produced by 
Escherichia coli bacteria into which has been inserted the human granulocyte 
colony-stimulating factor gene. Filgrastim is available as a sterile, clear, colorless, 
preservative-free liquid for parenteral administration at a specific activity of 1.0 ± 
0.6 x 10 U/mg (as measured by a cell mitogenesis assay). The product is available 
in single-use vials and prefilled syringes. The single-use vials contain either 300 µg 
or 480 µg filgrastim at a fill volume of 1.0 mL or 1.6 mL, respectively. It has been 
shown to be safe and effective in accelerating the recovery of neutrophil counts 
following a variety of chemotherapy regimens. Filgrastim is contraindicated in 
patients with known hypersensitivity to E. coli-derived proteins, or any component 
of the product. Allergic-type reactions occurring on initial or subsequent treatment 
have been reported in <1 in 4000 patients treated with filgrastim. These have 
generally been characterized by systemic symptoms involving at least 2 body 
systems, most often skin (rash, urticaria, facial edema), respiratory (wheezing, 
dyspnea), and cardiovascular (hypotension, tachycardia). Some reactions occurred 
on initial exposure. Reactions tended to occur within the first 30 minutes after 
administration IV. Rapid resolution of symptoms occurred in most cases after 
administration of antihistamines, steroids, bronchodilators, and/or epinephrine. 
Symptoms recurred in more than half the patients who were rechallenged. Other 
rarely reported serious complications associated with filgrastim include splenic 
rupture, acute respiratory distress syndrome, alveolar hemorrhage and hemoptysis, 
and sickle cell crises. G-CSF will be administered in clinic b trained personnel. 
 

 
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12.1.3 
Potential for Previously Unrecognized Drug Interactions 
Interferon alpha 2b has been co-administered with antiretroviral therapy in HIV-
infected individuals without any severe toxicity. Adverse events observed in 
HIV/HCV-coinfected participants receiving treatment with PEG INF and ribavirin 
were similar to those occurring in HCV-monoinfected participants. Drug interactions 
between antiretrovirals and treatment for hepatitis C concern mostly ribavirin and 
reverse transcriptase inhibitors (Mallolas and Laguno 2008). 
 
12.1.4 
Potential for Adverse Effects from Leukapheresis 
Risks associated with leukapheresis are similar to those seen with whole blood 
transfusions. They rarely include infection (<1% of persons) and commonly include 
nausea, fainting, dizziness, bruising at the needle puncture site, blood loss, and a 
transient decrease in platelet or red blood cell counts in 10% to 25% of persons. 
Potential problems of a leukapheresis procedure when citrate is given intravenously 
as an anticoagulant rarely includes seizures in <1% of persons and commonly 
includes muscle cramping, numbness, chilling, tingling sensations, and feelings of 
anxiety. Citrate is approved by the Food and Drug Administration for this use. If 
citrate/heparin or heparin alone is used as the anticoagulant, there may be a 
transient increase in clotting time, which may lead to blood loss in very rare 
instances (<1%). There is also a small risk of technical problems with the apheresis 
machine (1:1000 phereses). 
 
12.1.5 
Risks of Phlebotomy 
The primary risks of phlebotomy include occasional bleeding or bruising of the skin 
at the site of needle puncture, and the sensation of transient lightheadedness or 
rarely, fainting or infection. The amount of blood drawn for research purposes shall 
not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any 8-week period. 
 
12.2 Human Subject Protections 
Participants will be fully counseled prior to entry into the study as to the potential 
risks of the study. Participants who, in the opinion of the study team, do not fully 
comprehend these potential risks will not routinely be offered participation in the 
study. Participants will be monitored closely during their participation in the study, 
and informed of any new information regarding the study treatment that may 
influence their willingness to continue study participation. 
 
13 Study Recruitment  
The study will be advertised and posted on the NIH web site. The University of 
Pittsburgh AIDS Clinical Trials Unit will recruit participants from its Volunteer 
Registry. IRB-approved recruitment materials will be distributed among AIDS 
service organizations and other HIV providers in the region. Both the NIH and the 
University of Pittsburgh have recently completed drug intensification studies and 
have been successful in enrolling participants willing to complete the frequent visits. 

 
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In the most recent study, we used the current viral RNA enrollment criterion (≥0.38 
copies/mL) to study raltegravir intensification; 36 participants were screened for 
the study and 10 were eligible; the remainder were excluded chiefly on the basis of 
viral RNA levels. Nevertheless, 9 of the 10 enrolled participants completed this 
study and had evaluable data that were useful in determining whether viremia 
decreased. To improve our statistical analysis, we have increased the sensitivity of 
the assay (lower limit of quantification = 0.1 copies/ml) and will use 0.1 copies/ml 
as cutoff. 
 
13.1 Gender, Ethnicity, and Race Consideration 
Participants will not be excluded based on race, gender, or ethnicity. It is hoped 
that the racial and ethnic character of the participants in this study will reflect, as 
closely as possible, the demographics of the HIV epidemic in the United States. The 
NIAID has an outreach program to medically underserved minority participants in 
order to improve their access to clinical trials. The University of Pittsburgh’s AIDS 
Clinical Trials Unit makes a concerted effort to provide access to AIDS clinical trials 
for women and minorities. 
 
13.2 Children and Pregnant Women 
This study will be limited to adults 18 years of age and older. Insufficient data are 
available to evaluate the safety and efficacy of pegylated interferon in the pediatric 
population. In addition, this study is a no benefit study and would not be applicable 
to pediatric populations. The risks of pegylated interferon alpha 2b treatment 
during pregnancy have not been evaluated. Therefore, pregnant or nursing women 
will be excluded from this study. Any woman of childbearing potential must have a 
negative pregnancy test at screening, baseline, and prior to each dose of pegylated 
interferon and also agree to use two accepted methods of contraception throughout 
the study.  
 
 
14 Benefits/Compensation/Alternatives 
Financial compensation will be provided to study participants as outlined below. 
Participants will receive remuneration for the immediate costs associated with their 
study-related expenses like travel expenses, lodging, etc., as provided by the 
National Institutes of Health. 
 
Participants will be financially compensated according to the NIH Normal Volunteer 
Guidelines proportionate to the inconvenience of the procedures necessary to 
obtain the samples. The compensation schedule is as follows:  
Day 0 to Week 24: $40 at each visit  
Leukapheresis: $100 each 
 
The University of Pittsburgh AIDS Clinical Trials Unit will provide compensation for 
travel based on zip code and distance from the University. 
 

 
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This is a no benefit study; the alternative is not to participate. By participating in 
this study, participants may contribute to our understanding of persistent HIV 
viremia in individuals receiving potent antiretroviral therapy. 
 
15 Evaluation of Safety 
All enrolled participants who have received any study drugs will be evaluated for 
safety. Safety will be assessed by physical examination, vital signs, hematology, 
chemistry, urinalysis, and assessment of AEs. The severity of signs, symptoms, and 
AEs will be determined by using the Toxicity Severity Scale (Appendix 2). 
Recording/Documentation 
At each contact with the subject, information regarding adverse events will be elicited 
by appropriate questioning and examinations. All events, both expected/unexpected 
and related/unrelated will be recorded on a source document. Source documents will 
include: progress notes, laboratory reports, consult notes, phone call summaries, 
survey tools and data collection tools. Source documents will be reviewed in a timely 
manner by the research team. All reportable adverse events that are identified will 
be recorded in CRIMSON, or the appropriate case report form (CRF), or source 
document. The start date, the stop date, the severity of each reportable event, and 
the PI’s judgment of the AEs relationship and expectedness to the study 
agent/intervention will also be recorded in CRIMSON, or on the appropriate CRF, or 
source document. 
 
 
15.1 Protocol-specific Adverse Events  
15.1.1 
Definitions of Adverse Events 
Adverse Event: Any untoward or unfavorable medical occurrence in a human 
subject, that includes any abnormal sign (e.g. abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the subject’s 
participation in the research, whether or not considered related to the research. 
 
Serious adverse event: Any adverse event that 
• results in death; 
• is life-threatening (places the subject at immediate risk of death from the event as 
it occurred); 
• results in inpatient hospitalization or prolongation of existing hospitalization; 
• results in a persistent or significant incapacity; 
• results in a congenital anomaly/birth defect; or 
• based upon appropriate medical judgment, may jeopardize the subject’s health 
and may require medical or surgical intervention to prevent one of the other 
outcomes listed in this definition. (examples of such events include allergic 
bronchospasm requiring intensive treatment in the emergency room or at home, blood 
dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse) 
 

 
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Protocol Deviation: Any change, divergence, or departure from the IRB approved 
study procedures in a research protocol. Protocol deviations are designated as 
serious or non-serious and further characterized as 
 
1. Those that occur because a member of the research team deviates from the 
protocol. 
2. Those that are identified before they occur, but cannot be prevented. 
3. Those that are discovered after they occur 
 
Serious Protocol Deviation: A deviation that meets the definition of a Serious 
Adverse Event or compromises the safety, welfare or rights of subjects or others. 
 
Non-compliance: The failure to comply with applicable NIH HRPP  policies, IRB 
requirements, or regulatory requirements for the protection of human subjects. 
Non-compliance is further characterized as 
1. Serious: Non-compliance that 
a. Increases risks, or causes harm, to participants 
b. Decreases potential benefits to participants 
c. Compromises the integrity of the NIH-HRPP 
d. Invalidates the study data 
2. Continuing: Non-compliance that is recurring 
3. Minor: Non-compliance that, is neither serious nor continuing. 
Unanticipated Problem (UP): Any incident, experience, or outcome that meets all 
three of the following criteria would be considered a serious UP:  
 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol 
and informed consent document; Investigator’s Brochure or other study 
documents; and 
b. the characteristics of the subject population being studied 
2. related or possibly related to participation in the research 
3. suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized. 
 
Unanticipated problem that is not an Adverse Event (UPnonAE): An unanticipated 
problem that does not fit the definition of an adverse event, but which may, in the 
opinion of the investigator, involve risk to the subject, affect others in the research 
study, or significantly impact the integrity of research data.  These events may 
involve a greater risk of social or economic harm to subjects or others rather than 
physical/psychological harm. Such events would be considered a non-serious UP. 
Examples of a UPnonAE include a breach of confidentiality, accidental destruction of 
study records, or unaccounted-for study drug. 
 

 
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15.1.2 
Adverse Event Reporting 
Expected adverse events are those listed in the package inserts for Pegintron. 
Adverse events will be graded according to the Division of AIDS Table for Grading 
Adults Adverse Events (http://rcc.tech-res.com/safetyandpharmacovigilance/ , 
Appendix 2), assessed for severity (mild/moderate/severe), expectedness 
(expected/unexpected), and relatedness to study drug (definitely, probably, 
possibly, unlikely, or unrelated). All adverse events will be recorded in the study 
database. Serious adverse events will be reported to the participating IRBs and the 
FDA. Adverse events that do not meet guidelines for expedited reporting will be 
included with each continuing review. 
 
15.1.2.1 
 
Expedited Reporting to the NIAID IRB 
 
Serious and non-serious Unanticipated Problems, deaths, serious deviations, and 
serious or continuing non-compliance will be reported within 7 calendar days of 
investigator awareness. Serious Adverse Events that are possibly, probably, or 
definitely related to the research will be reported to the NIAID IRB within 7 calendar 
days of investigator’s awareness, regardless of expectedness. 
15.1.2.2 Waiver of Reporting Anticipated Protocol Deviations, Expected 
non-UP AEs and Deaths   
Anticipated deviations in the conduct of the protocol will not be reported to the IRB 
unless they occur at a rate greater than anticipated by the study team. Expected 
adverse events will not be reported to the IRB unless they occur at a rate greater 
than that known to occur in HIV infection.  If the rate of these events exceeds the 
rate expected by the study team, the events will be classified and reported as 
though they are unanticipated problems. Deaths related to the natural history of 
HIV infection will be reported at the time of continuing review. 
 
15.1.2.2 
Annual Reporting 
The following items will be reported to the NIAID IRB in summary at the time of 
Continuing Review: 
 
- 
Serious and non-serious unanticipated problems  
- 
Expected serious adverse events that are possibly, probably, or definitely 
related to the research 
- 
Serious adverse events that are not related to the research 
- 
All adverse events, except expected AEs and deaths granted a waiver of 
reporting.  
- 
Serious and Non-Serious Protocol deviations  
- 
Serious, continuing, and minor non-compliance 
- 
Any trends or events which in the opinion of the investigator should be 
reported 
- 
Protocol specific reporting as applicable 

 
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- 
 
15.1.2.3 
Reporting of Pregnancy  
Pregnancy that occurs during the course of the protocol will be reported within 7 
days of the investigator’s awareness 
15.1.2.4         Type and Duration of the Follow-up of Subjects after Adverse 
Events 
Serious adverse events will be followed until resolution; Some adverse events resulting 
that do not resolve completely will be followed until stable.  Pregnancy will be followed 
until 3 months after delivery, miscarriage, or termination. 
 
16 Monitoring 
This is an open-label treatment protocol. Monitoring will be performed by the site 
investigators. The NIAID and the University of Pittsburgh Institutional Review 
Boards will review the study at least annually. Because of the nature of the study, 
we plan to constitute a Data Safety Monitoring Board (DSMB) at the NIH to review 
the study progress and safety at least annually.  This function will be provided 
through NIAID DSMB, although the group will be functioning as a safety monitoring 
committee. 
 
The NIAID Intramural DSMB is constituted to review the safety data of Intramural 
NIAID clinical studies that require DSMB oversight, and consists of experts in 
infectious diseases, biostatistics, and clinical trials. The DSMB will meet at least 
once a year to review the completeness of the study data collected, the adherence 
to the protocol, and PI’s review summaries to evaluate safety, study conduct, and 
scientific validity and integrity of the study. Other reviews may occur as needed if 
other safety issues arise. 
 
The initial review will occur prior to opening the study to enrollment. 
16.1 Stored Samples and Future Research 
Extra blood samples will be stored using a code number that only the study team 
can trace back to the subject.  These samples will be stored for other investigators 
who may want to pursue additional research using these stored samples. If so, the 
NIH study team may send these samples to them, along with the coded number 
label. Once the protocol is terminated and data analysis is completed remaining 
samples will be maintained for future research by appropriate transfer to other 
protocols for ongoing human subjects protection oversight; requesting the use of 
stored samples may be submitted to the IRB for approval if the intended use is not 
covered by original consent. Any loss or destruction of stored samples will require 
written IRB notification.  
 
16.2 Data Management Plan 
All research data and results will be carefully recorded using data collection forms 
that will be saved and allow for continuous access. Further, all collected data will be 
entered and saved to a password-protected computer spreadsheet (Microsoft 

 
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ExcelTM) for analysis and review. The investigator is responsible for assuring that 
the data collected is complete, accurate and recorded in a timely manner.  Source 
documentation should support the data collected on the CRF and must be signed 
and dated by the person recording and/or reviewing the data.  All data will be 
annotated and indexed, including all notebooks and computerized information, to 
facilitate detailed review of data. All data, even those of experiments not directly 
leading to publication, will be treated comparably. All research data will be made 
available to scientific collaborators and supervisors for immediate review, consistent 
with requirements of confidentiality.   All research data, including the primary 
experimental results, will be retained for a minimum of 5 years to allow for analysis 
and repetition by others of published material resulting from the data.  
 
Demographic information and laboratory results will be collected using case report 
forms and then transcribed into an electronic database on an ongoing basis as 
participants are enrolled. At the NIH site, data will be stored in the CRIMSON 
database.  Other experimental data, including HIV viral load results and IFN drug 
levels, will be analyzed and reported in batches. Standard laboratory results will be 
accessed by the site investigators and the study coordinators. All results, whether 
recorded on paper or electronically, will be maintained using only the subject's 
numeric code for identification. Data collection and quality assurance will be 
performed primarily by the site investigators and study coordinators. 
 
Data management, including the decision to publish, will be the responsibility of the 
Co-Principal Investigators. After publication, all research data that form the basis of 
that communication will be made available promptly and completely to responsible 
scientists seeking further information. Exceptions include those requests that would 
infringe on confidentiality of clinical data or if unique materials were obtained under 
agreements that preclude their dissemination. 
 
17 Anonymity and Confidentiality 
As each subject is consented and then enrolled, he or she will be allocated a unique 
study number. To ensure subject confidentiality, these numeric codes will substitute 
for personal identifiers on all paper documents, computer records, and blood 
sample vials. The information obtained during the conduct of this clinical study is 
confidential, and disclosure to third parties other than those noted is prohibited. 
The results of the research study may be published, but subject’s names or 
identities will not be revealed. Records will remain confidential. To maintain 
confidentiality, the Principal Investigators will keep records with personal identifiers 
in double-locked cabinets and the results of tests will be coded to prevent 
association with subject names. It is expected that this data will be reported in 
scientific journals and scientific meetings. Confidentiality of participants will be 
maintained in all forms of reporting. Participants will be informed in general terms 
of the results as soon as practical.  
 
 

 
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Visit
Screen
Pre-Ent 1
Pre-Ent 2
Entry
1
2
3
4
5
6
7
8
9
10
11
12
Final Visit*
Day/Wk
D-58 to -28
D-27 to -1
D-14 to -1
D0
D7
D14
D21
D28
D35
D42
D49
D56
D84
W16
W24
W36
W48
Consent
x
x
Physical
x
x
Vital signs
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Tar. Phys.
x
x
x
x
Med Hx
x
Con meds
x
x
BDS
x
x
x
x
x
x
x
x
x
x
x
x
x
Psy Eval
**
**
C/D/Pl
x
x
x
x
x
x
x
x
x
x
x
x
x
Chem*
x
x
x
x
x
x
x
x
x
x
x
x
x
FBS 
X
ALT,Bili
x
x
x
x
x
x
x
x
x
x
x
x
x
Amylase
x
Creat Cl
x
x
x
x
x
x
x
x
x
TSH/FT4
x
UA
x
Preg
x
x
x
x
x
ELISA/WB**
x
ANA
x
Hep B SA
x
HepB Sab
x
HepC Ab
x
HepC VL
x
CD4+
x
x
x
x
x
x
HIV RNA
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
SCA
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Eye exam
x
PBMC 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Apheresis
x
x
IFN admin
x
x
x
x
IFN level
x
x
x
x
x
*Chem&includes&Creatinine,&lytes,&BUN,&Alk&phos
&&&&&&&&&&&&&&**
* If not available 
&

 
IFN-alpha2b for HIV on ARV (PITHRA), Version 4.0 
 
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Appendix 2: Toxicity Severity Scale 
The toxicity grading scale for this protocol is the “Table for Grading the Severity of Adult and Pediatric 
Adverse Events; Version 1.0 - December 2004 (Clarification dated August 2009).”    
 
It can be found at: 
http://rcc.tech-res.com/safetyandpharmacovigilance/