CD4 T CELL ASSESSMENTS IN EVALUATION OF HIV THERAPEUTICS - Flow Cytometry in Drug Discovery and Development - page 177

HAART has shown not to be as effective in patients with advanced disease;
therefore, stage of infection has a critical role in howwell ARV therapywill work [36].
Studies have shown that patients who have immune systems that are capable of
controlling the virus and preventing opportunistic infections may exhibit a healthier
prognosis when on HAART [25]. Therapy must be initiated before the patient's
immune system is too compromised to effectively reconstitute CD4 T cells [36].
Routine monitoring of CD4 counts can anticipate progression to AIDS and can also
detect drug resistance allowing the initiation of therapy or changing to alternative
ARV therapeutics to combat resistance. HIV-infected patients should have CD4 levels
routinely checked one to two times per year. In resource-limited settings where CD4
testing is not readily available or is too expensive, clearer evidence of immunosup-
pression may be needed before treatment can be recommended. Complications from
long-term use of HAART are still unknown; however, studies have shown that
delaying treatment has long-term disadvantages in terms of patient survival [37].
Studies have shown that patients who were severely immunodeficient at the start of
therapy experienced higher rates of AIDS and death up to 6 years later [37]. In
addition, there is increasing evidence to suggest that there is an increased risk of
resistance to HAART if treatment is started with a low CD4 count. A recent study
found that the frequency of HIV resistance mutations was 50% among patients who
started HAART with a CD4 T cell count between 0 and 349 cells/mm 3 compared to
a frequency of 22% among patients who started HAART at a CD4 T cell count

350 cells/mm 3 [38]. This finding suggests that initiating HAARTat higher CD4 cell
counts may decrease the risk of developing resistance to therapy.

8.5 EVOLUTION OF CD4 TESTING AND METHODOLOGY

Over the past 25 years, there have been major technological advances in the way that
CD4 þ T cells are enumerated. The utility of CD4 measurements as a prognostic
marker for HIVinfectionwas an early finding in the understanding of HIV/AIDS [39].
The technology available to perform this testing included flow cytometry as well as
manual counting by light and fluorescent microscopy. These experiments rely on the
use of monoclonal antibodies targeted to the CD4 antigen on T lymphocytes. The CD4
molecule is a transmembrane glycoprotein approximately 55 kDa in size that is
involved in cell activation upon its recognition to target antigen-presenting cells [40].
CD4 positive T cells are selected during their maturation from hematopoietic stem
cells in the bone marrow to peripheral blood circulation via the thymus, by their
affinity toMHC II complexes [41, 42]. Production of antibody to the CD4 molecule in
the late 1970s led to further identification of the involvement of the T helper cell
population in cellular immunity. Variability in antibody specificity led to the strategy
of using antibody combinations to exclude non-T cells in the analysis. Prior to antigen
nomenclature conventions, the OKT4 antibody clone developed byOrthoDiagnostics
(Ortho Clinical Diagnostics, Rochester, NY), T4 clone by Coulter Corporation
(Beckman Coulter, Inc., Miami, FL) and Leu-3 clone by Becton Dickinson {BD
Biosciences, San Jose, CA] were the first antibodies widely used for CD4 cell