ABSTRACT
The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay.
Subject(s)
AIDS Vaccines/immunology , Flow Cytometry , HIV Antibodies/immunology , High-Throughput Screening Assays , AIDS Vaccines/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , HIV Core Protein p24/immunology , HIV Envelope Protein gp120/immunology , HeLa Cells , Humans , Jurkat Cells , Sensitivity and SpecificityABSTRACT
Cooperation between endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. We developed a unique in vitro tissue-engineered model to study angiogenesis. The human endothelialized reconstructed connective tissue model promotes the formation of a three-dimensional branching network of capillary-like tubes (CLT) with closed lumens. The purpose of this work was to investigate whether pericytes were spontaneously recruited around CLT in the model. We demonstrated that smooth muscle α-actin (SMA)-positive cells were found closely associated with PECAM-1-positive capillaries in the model. Twelve percent (±2.6) of SMA-positive cells were detected along with 15% (±1.64) von Willebrand factor-positive endothelial cells in the culture system after 31 days of in vitro maturation. Conversely, no SMA-positive cells were detected in reconstructed connective tissues made solely of fibroblasts. Knowing that PDGF is a major factor in the recruitment of pericytes, we showed that blockade of the PDGFB receptor using the inhibitor AG1296 induced an overall 5, 2.6, and 2.4-fold decrease in the SMA-positive cells, von Willebrand factor-positive cells, and number of capillaries, respectively. Using combinations of human GFP-positive fibroblasts and endothelial cells, we demonstrated that pericytes were recruited from the fibroblast population in the model. In conclusion, our tissue-engineered culture system promotes the spontaneous formation of a network of capillaries and the recruitment of pericytes derived from fibroblasts. Since pericytes are essential components of the blood microvasculature, this culture system is a powerful model to study angiogenesis and endothelial cell/pericyte interactions in vitro.
Subject(s)
Fibroblasts/cytology , Fibroblasts/physiology , Neovascularization, Physiologic/physiology , Pericytes/cytology , Pericytes/physiology , Tissue Engineering/methods , Actins/metabolism , Becaplermin , Capillaries/cytology , Capillaries/physiology , Cell Culture Techniques , Human Umbilical Vein Endothelial Cells , Humans , Infant, Newborn , Models, Cardiovascular , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Proto-Oncogene Proteins c-sis/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , von Willebrand Factor/metabolismABSTRACT
Local breast radiation therapy (RT) is associated with a 3-fold increased risk of secondary acute myeloid leukemia. As a first step in determining the mechanism(s) underlying this observation, we investigated the role of RT in mediating the active recruitment of hematopoietic stem cells (HSC) to the site of RT. Our results show in a mouse model that local RT delivered to the left leg causes preferential accumulation of bone marrow mononuclear cells to the irradiated site, with maximum signal intensity observed at 7 days post-RT. This is associated with a 4-fold higher number of donor-derived HSC present in the left leg, demonstrating recruitment of HSC to the site of RT. SDF-1, matrix metalloproteinase 2 (MMP-2), and MMP-9 expression is significantly increased in the irradiated bone marrow, and their inhibition significantly reduced HSC recruitment to the irradiated bone marrow. Our data show that local RT has significant systemic effects by recruiting HSC to the irradiated bone marrow site, a process mediated by SDF-1, MMP-2, and MMP-9. These results raise the possibility that the exposure of increased numbers of HSC at a local site to fractionated irradiation may increase the risk of leukemogenesis. Our data also suggest some opportunities for leukemia prevention in breast cancer patients undergoing RT.
