Evaluation of a Blood Test to Measure Immune Function in HIV Positive People Compared With HIV Negative People

Routine virologic and immunologic tests to monitor HIV-1 in infected individuals on highly
active antiretroviral therapy (HAART) include CD4 T-cell enumeration, HIV-1 plasma viral
load, and HIV-1 genotypic resistance testing at baseline for treatment-naive patients and
following virologic failure during HAART.

The CD4 T-cell count is a measurement of the number of T helper lymphocytes and, in
conjunction with the CD4/cluster of differentiation 8 (CD8) ratio, are surrogate markers to
evaluate the status of the immune system and used to monitor the progression and define
stages of HIV disease in infected subjects either on or off treatment. An increasing or high
CD4 count and CD4/CD8 ratio between 1 and 4 suggests control of virus replication as a
result of HAART (or from inherent immune activity in untreated subjects) while a decreasing
or low CD4 count and a CD4/CD8 ratio <1 suggests HAART failure and/or a deteriorating immune
system.

HIV-1 plasma viral load measures the number of HIV-1 RNA copies derived from virus particles
in plasma. A detectable plasma viral load indicates HIV-1 replication while an undetectable
plasma viral load suggests control of HIV-1 replication. Nevertheless, with the lower limit
of detection of 50 copies HIV-1 RNA/mL for commercial assays, lower levels of plasma virus
can remain undetected by these assays but detectable in approximately 70% patients on HAART
using research assays with a cutoff of <50 copies HIV-1 RNA/ml. Such research based plasma
viral load assays are never used for routine monitoring in the clinical care of infected
subjects.

While CD4 counts and HIV-1 plasma viral load are inextricably related in the context of
HIV-1 disease and currently the approved tests to determine HIV-1 response to HAART these
assay have significant shortcoming for the effective treatment of HIV as defined below:

- CD4 counts and immune dysfunction. Increasing or high CD4 counts or normal CD4/CD8
ratios as a result of HAART may not necessarily represent an uncompromised immune
system but can represent a state of immune dysfunction as a result of immune activation
from continued HIV replication.

- HIV Cellular reservoirs: Low levels of cell associated HIV replication undetected in
plasma by standard plasma viral load assay. Cell associated HIV replication is
determined by HIV polymerase chain reaction (PCR) from extracted and purified total
cell associated RNA or in-situ hybridization to cell associated HIV-1 RNA in intact
cells, and detected in approximately 62% to 80% patients on HAART with a plasma viral
load of <50 copies HIV-1 RNA/mL. Targeted treatment, cell associated HIV drug efficacy,
cell associated HIV resistance and identification of the cellular source of HIV can be
determined by HIV cell reservoirs assays and not by plasma viral load.

- Functional immune response differences based on cell associated HIV-1 transcriptional
activity and not undetectable plasma viral load. Immune responses in patients on HAART
with chronic, low level HIV replication in circulating T lymphocytes and undetectable
plasma viral load include significant in-vitro proliferation to HIV-1 p24 antigen, no
significant response to recall antigen tetanus toxoid and higher responses to pathogen
antigens compared to patients on HAART with undetectable HIV replication in memory T
lymphocytes and undetectable plasma viral load.

- Adherence of plasma derived HIV-1 virions to erythrocytes. Erythrocytes sequester
plasma associated HIV-1 virions in whole blood from some patients on HAART. While these
patients have undetectable plasma viral load, the corresponding whole blood has viral
load values ranging from 234 to 82,878 copies HIV-1 RNA/mL whole blood. These patients
with detectable whole blood viral load but with undetectable plasma viral load display
clinically advanced HIV infection.

- CD4 counts and inexplicable response to HAART. A decrease in CD4 counts with an
undetectable plasma viral load may be explained by the sequestering of HIV plasma virus
to erythrocytes or low levels of cell associated HIV replication.

- Defective/non-infectious virions: HIV-1 plasma viral load assays cannot be used to
differentiate between infectious and non-infectious or defective virus particles as
<0.2 % circulating HIV-1 virions (or from 1:477 to 1: 117,803) are infectious.
Consequently, those genotypic and phenotypic drug resistance assays, and HIV-1 tropism
assays that utilize HIV-1 RNA derived from plasma virus HIV-1 RNA will generate results
from mostly non-infectious or defective virus. Difference in nucleic acid sequences
between infectious and non-infectious or defective virus has not been appropriately
investigated in relation to diagnostic and monitoring tests. The implications of
utilizing viral sequences from predominantly non-infectious or defective viral
particles opposed to infectious virus particles for drug resistance and tropism tests
in the clinical care of HIV infected patients is unknown.

Consequently, increasing evidence demonstrates that CD4 counts and plasma viral load cannot
be used to fully interpret the response of HIV to HAART. Moreover, mounting data proves that
continued HIV-1 replication in the absence of detectable plasma viral load generates HIV and
viral antigen resulting in immune activation thereby enabling HIV pathogenesis as
demonstrated from increased levels of T cell turnover and proliferation, apoptosis of
uninfected T cells as well as polyclonal B cell, natural killer (NK) cell and monocyte
activation. Various cell based markers have been associated with HIV pathogenesis including
immune activation markers Human Leukocyte Antigen (HLA)-Cell Surface Receptor (DR) (HLA-DR),
cluster of differentiation 38 (CD38), cluster of differentiation 69 (CD69), cluster of
differentiation 71 (CD71), senescence marker cluster of differentiation 57 (CD57),
proliferation marker Ki67 and apoptosis marker cluster of differentiation 95 (CD95) (FasR).
Altogether these markers of HIV pathogenesis enable inflammatory responses, reseeding
reservoirs, HIV-1 evolution and drug resistance, and non-opportunistic diseases with a
gradual deterioration of the immune system.

Immune function tests evaluate the functionality of a component of the immune system. They
are generally in-vitro based assays and are designed to measure an outcome following
exposure of lymphocytes to specific antigens or mitogens. The outcome may be the
measurement of an immune stimulation marker such as secretion of gamma interferon,
incorporation of tritiated thymidine into cellular genomic DNA as a result of a
lymphoproliferative cell response to recall antigen and/or mitogen or, in the case of the
in-vivo based Tuberculin Skin Test, the size of skin induration at the site of tuberculin
purified protein derivative administration.

Cellestis Limited - A Qiagen Company, has recently developed the QuantiFERON® Monitor (QFM
or CST007) test based on the patented QuantiFERON® Technology to provide a measure of the
cell-mediated immune function. It is an in vitro diagnostic test that uses a combination of
stimulants to specifically stimulate different immune cells in the whole blood and measures
the interferon-gamma released in the plasma by ELISA (Enzyme-linked immunosorbent assay).
Therefore, the aims of this study will address the utility of QFM in the HIV
immunocompromised setting and as cell associated markers HIV pathogenesis influence immune
function the HIV Viral Reservoir assay blood test will be included to better understand the
level of immune function within and between cohorts.

How long will it take? One visit for about 1 hour with Dr. Gatpolintan and his Clinical
Study Coordinator to answer questions, then about 10 minutes for a blood draw (nine blocks
from Dr. Gatpolintan' office).

Study outcome measures (Correlation between QFM and CD4 counts and CD4/CD8 ratios) will be
assessed, including data presentation, within an average period of 1 year after study
subject enrollment.

Inclusion Criteria:

Anti-retroviral drug naïve, (never on treatment, or >60 days off treatment), n=30

- All plasma viral load results within the last 24 months; most recent plasma viral
load result of any value used for enrollment.

- All CD4 results within the last 24 months; most recent CD4 result used for
enrollment: CD4>500/microliter (uL) (n=15) or CD4<500/uL (n=15).

Successful HAART > 24 months with two undetectable plasma viral load within the last 12
months, n=30

- All plasma viral load results within the last 24 months; two most recent plasma viral
load results within last 12 months must be undetectable and used for enrollment.

- All CD4 results within the last 24 months; most recent CD4 result used for
enrollment: CD4>500/uL (n=15) or CD4<500/uL (n=15).

On HAART > 24 months with latest plasma viral load >200, n=30

- All plasma viral load results within the last 24 months; most recent plasma viral
load result >200 and used for enrollment.

- All CD4 results within the last 24 months; most recent CD4 result used for
enrollment: CD4>500/uL (n=15) or CD4<500/uL (n=15).

HIV Uninfected Controls (n=30):

- Documentation of HIV seronegative status at time of enrollment

Exclusion Criteria:

Key Exclusion Criteria for all HIV Infected only:

- Primary infection: < 6 months after documented HIV-1 antibody positive test

Key Exclusion Criteria for drug naïve HIV Infected only

- Ended HIV medications less than 2 months before the study

Key Exclusion Criteria for HIV uninfected only:

- On HIV-1 pre-or post exposure prophylaxis <21 days before enrolment

Key Exclusion Criteria for all subjects:

- <18 or >65 years of age

- Pregnant or lactating subjects

- Documented hepatitis B virus (HBV) and/or hepatitis C virus (HCV) Infection

- Proven or suspected acute hepatitis

- Transient clinical manifestation (i.e., cold, flu, measles, etc). Eligible when
resolved

- Evidence of a gastrointestinal malabsorption syndrome, chronic inflammatory disease
(i.e. Crohn's Disease) or chronic nausea or vomiting

- Prior history of significant renal or bone disease

- Malignancy other than cutaneous Kaposi's sarcoma or basal cell carcinoma

- < 30 days after any vaccination. Eligible 30 days post vaccination.

- Current alcohol or substance abuse

- Active, serious infections (other than HIV infection) requiring parenteral
antimicrobial therapy within 30 days prior to enrollment.

- Any other clinical condition in the opinion of the PI, would make the subject
unsuitable for the study i.e. active cytomegalovirus (CMV) and Epstein-Barr virus
(EBV) infection, diabetes, Rheumatoid Arthritis, etc.

- Previous therapy with agents with systemic myelosuppressive, pancreotoxic,
hepatotoxic or cytotoxic potential within 3 months of study start or the expected
need for such therapy at the time of enrollment

- On therapy that suppresses bone marrow, toxic to liver and pancreas

- On ongoing therapy that is toxic to kidneys including aminoglycoside antibiotics,
amphotericin B, cidofovir, cisplatin, foscarnet, IV pentamidine, other agents with
significant nephrotoxic potential

- Creatinine clearance < 60 mL/min

- On anti-cancer therapy

- On ongoing therapy with immunosuppressive agents

- On ongoing chemotherapeutic agents

- On ongoing systemic corticosteroids

- On ongoing systemic interleukin 2 or other cytokine therapy

- Anticonvulsants (eg. Carbamazepine, Phenytoin, Valproate)

- Monoclonal antibody therapy (eg. Muromonab OKT3)

- Any other prior therapy that, in the opinion of the PI, would make the subject
unsuitable for the study.

How long will it take? One visit for about 1 hour with Dr. Gatpolintan and his Clinical
Study Coordinator to answer questions, then about 10 minutes for a blood draw (nine blocks
from Dr. Gatpolintan' office).