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.