Therapeutic immunization in HIV infected Ugandans receiving stable antiretroviral treatment: a Phase I safety study

Therapeutic immunizations in HIV infection may boost immunity during antiretroviral treatment. We report on the first therapeutic vaccine trial in Uganda, Africa. This open label Phase I trial was designed to assess the safety, tolerability and immunogenicity of a therapeutic HIV-1 vaccine candidate. Thirty HIV positive volunteers receiving a stable regimen of antiretroviral therapy with CD4 counts >400 were recruited for the safety evaluation of LFn-p24C, a detoxified anthrax-derived polypeptide fused to the subtype C HIV gag protein p24. The vaccine was well tolerated and HIV RNA levels remained undetectable following three immunizations. CD4 counts in vaccine recipients were significantly higher compared to the control individuals after 12 months. HIV-specific responses were associated with higher gain in CD4 counts following LFn-p24C immunizations. Volunteers were subsequently asked to undergo a 30-day period of observed treatment interruption. 8/24 (30%) individuals showed no evidence of viral rebound during treatment interruption. All demonstrated prompt suppression of viral load following resumption of ART. Our data demonstrate the safety of LFn-p24C and suggest that adjunct therapeutic immunization may benefit select individuals in further boosting an immune response.     

Quality Monitoring of HIV-1-Infected and Uninfected Peripheral Blood Mononuclear Cell Samples in a Resource-Limited Setting

Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependent on the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with a high level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function of PBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility and sample quality at the sites that perform cryopreservation. We have developed a comprehensive internal quality control program to monitor processing, including components of method validation. Quality indicators for real-time performance assessment included the time from venipuncture to cryopreservation, time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immune phenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for both HIV-1-infected and uninfected subjects (P < 0.007), but all other major lymphocyte subsets were unchanged. Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background and all specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2 production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decay with regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it is possible to achieve the high level of quality necessary for vaccine trials and natural history studies in a resource-limited setting and provide strategies for laboratories to monitor PBMC processing performance.Cryopreserved peripheral blood mononuclear cells (PBMC) are critical to studies of HIV-1 infection and for the development of an effective HIV vaccine. High-quality cryopreservation is particularly challenging and important in resource-limited settings, where natural history, therapeutic, and preventative protocols are being developed. Cryopreservation and batch testing allow for simultaneous assessment of critical samples, thereby reducing interassay variability. Since standard method validation measures, such as accuracy, precision, linearity, and reportable ranges, are not easily obtained for PBMC processing, more studies are needed to understand the parameters influencing the reproducibility of processing and quality of cryopreserved PBMC samples in resource-limited settings.Separation of PBMC should yield a pure population of mononuclear cells (95% ± 5%) (18), with minimal contamination from red blood cells, granulocytes, and platelets. Anticipated yields of mononuclear cells from healthy donors are approximately 1 × 10⁶ to 2 × 10⁶ cells/ml of whole blood, with a purity of 60 to 70% lymphocytes at >95% viability, with a reduction of platelets to <0.5% of the original whole-blood content (25). However, the data on the impact of cryopreservation on lymphocyte phenotypes are inconsistent (4, 31, 34, 39, 41). Additionally, there are differences in hematologic parameters and lymphocyte subsets in African populations compared to populations in the industrialized world (17, 26, 33, 44). Careful examination of the impact of cryopreservation on yield and phenotype is therefore needed for this region. Furthermore, studies indicate an increased ability to preserve the function of cryopreserved PBMC compared to cells in whole blood, as measured by proliferation assays (1, 12, 23, 46, 47), cytokine production (9, 28, 30, 32, 37), apoptosis (40), and HLA tetramer staining (2). This is particularly important for HIV-1, as many vaccine candidates are designed to elicit T-cell responses, and reliable PBMC-based assays such as enzyme-linked immunospot (ELISPOT) assay and multiparameter flow cytometry are required to prioritize candidates for large-scale efficacy testing.Some studies indicate that the functional integrity of cryopreserved PBMC samples is maintained (22), while other reports suggest a loss of function (38). Preanalytic variables which have been examined closely include time from blood draw to initiation of processing, type of anticoagulant, proper mixing of blood tubes, and transport temperature (9, 13, 28, 45). Analytic variables that impact PBMC processing include separation method, type of cryoprotectant medium, prechilling of the freeze containers, cryopreservation parameters, and temperature of the thawing medium (9, 15, 28, 30, 37). In addition to defining processing parameters, it is important to monitor quality assurance for cryopreserved PBMC assays after cryopreservation (7). However, few studies have addressed the importance of continuous monitoring of the processing and cryopreservation of PBMC themselves but have focused instead on the procedures for thawing of cells in labs that perform end-point assays. We have developed a comprehensive internal quality control program to monitor PBMC processing. This study describes this program and the results achieved in a resource-limited setting. Additionally, we describe procedures to evaluate our laboratory methods for PBMC processing.