Publications

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-gamma responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4(+) T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8(+) T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8(+) T cell responses to peptides and peptide pools were well preserved. Loss of both CD4(+) and CD8(+) T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4(+) T cell responses to peptide stimulation and partially restored T cell IFN-gamma responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-gamma responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4(+) T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4(+) and CD8(+) T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4(+) T cell responses and mild skewing of T cell phenotypic marker expression.

BACKGROUND

The SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals.

METHODOLOGY/PRINCIPAL FINDINGS

Endpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions.

CONCLUSION

Given the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans.

T-cell activation has long been considered a central mediator of HIV pathogenesis. High T-cell activation levels predict more rapid disease progression in untreated patients and decreased treatment-mediated CD4+ T-cell gains during antiretroviral therapy, independent of plasma HIV RNA levels, and may be the primary feature distinguishing pathogenic from nonpathogenic primate models of HIV infection. Studies in animal models and individuals with HIV infection continue to enhance our understanding of how T-cell activation causes immunodeficiency during HIV infection. The goal of these studies is to identify specific mechanisms that can be targeted by novel immune-based therapies for patients who have thus far been unable to recover normal immune function despite years of antiretroviral therapy. Although most immune-based therapies targeting T-cell activation have been unsuccessful to date, recent scientific developments have focused attention on specific pathways that may be exploited by future generations of immune-based therapies.

Four new compounds, belonging to the tetranortriterpenoid family, named dysoxylins A-D (1-4), isolated from Dysoxylum gaudichaudianum, were found to exhibit potent antiviral activity against respiratory syncytial virus (RSV). These structures were determined by NMR spectroscopy and mass spectrometry and were shown to have anti-RSV EC50 activities in the range 1.0-4.0 microg/mL in cytopathic effect inhibition and plaque reduction assays.