Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells.

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 µg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 10(6) PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/10(6) PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.

Generation of immunogenic dendritic cells from human embryonic stem cells without serum and feeder cells.

AIM: Dendritic cell (DC)-based vaccines have a potential utility for use in the treatment of malignancy. Human embryonic stem cells (hESCs) may provide a more cost-effective and reliable source of DCs for immunotherapy purposes, providing on-demand access for patients. METHOD: We developed a protocol to generate DCs from hESCs in vitro in the absence of serum and feeder cells. This protocol uses growth factors bone morphogenetic protein-4, granulocyte macrophage-colony stimulating factor (GM-CSF), stem cell factor and VEGF in serum-free media to generate hESC-derived monocytic cells. These cells are further differentiated to hESC-derived immature DCs with GM-CSF and IL-4, and matured to hESC-derived mature DCs with a maturation cocktail consisting of GM-CSF, TNF-alpha, IL-1beta, IFN-gamma and PGE2. RESULTS: This study demonstrates the applicability of our defined differentiation process in generating functional hESC-derived DCs from multiple hESC lines. We show that hESC-derived immature DCs phagocytose, process, and present antigen upon maturation. hESC-derived mature DCs express the maturation marker CD83, produce Th1-directing cytokine IL-12p70, migrate in response to chemokine, and activate both viral and tumor antigen-specific T-cell responses. CONCLUSION: We developed a chemically defined system to generate unlimited numbers of DCs from hESCs. Our results demonstrate that hESC-derived DCs generated from this process are immunogenic and have the potential to be used for DC immunotherapy.

Directed evolution of gene-shuffled IFN-alpha molecules with activity profiles tailored for treatment of chronic viral diseases.

Type I IFNs are unusually pleiotropic cytokines that bind to a single heterodimeric receptor and have potent antiviral, antiproliferative, and immune modulatory activities. The diverse effects of the type I IFNs are of differential therapeutic importance; in cancer therapy, an enhanced antiproliferative effect may be beneficial, whereas in the therapy of viral infections (such as hepatitis B and hepatitis C), the antiproliferative effects lead to dose limiting bone marrow suppression. Studies have shown that various members of the natural IFN-alpha family and engineered variants, such as IFN-con1, vary in the ratios between various IFN-mediated cellular activities. We used DNA shuffling to explore and confirm the hypothesis that one could simultaneously increase the antiviral and Th1-inducing activity and decrease the antiproliferative activity. We report IFN-alpha hybrids wherein the ratio of antiviral:antiproliferative and Th1-inducing: antiproliferative potencies are markedly increased with respsect to IFN-con1 (75- and 80-fold, respectively). A four-residue motif that overlaps with the IFNAR1 binding site and is derived by cross breeding with a pseudogene contributes significantly to this phenotype. These IFN-alphas have an activity profile that may result in an improved therapeutic index and, consequently, better clinical efficacy for the treatment of chronic viral diseases such as hepatitis B virus, human papilloma virus, HIV, or chronic hepatitis C.

A chimeric tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques.

DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general.

Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters.

Genetic vaccinations, gene therapy, and manufacturing of therapeutic proteins would benefit from promoter sequences that provide improved or prolonged expression levels. The cytomegalovirus (CMV) promoter is one of the most potent promoters known to date, and no previous examples of improved activity of this promoter by sequence mutagenesis have been reported. This study describes directed molecular evolution of CMV promoters derived from two human and two nonhuman primate strains of CMV by DNA shuffling and screening. Libraries of chimeric promoters were screened and analyzed for expression levels and immune responses, using plasmid DNA vectors encoding luciferase and beta-galactosidase. The results indicate that high functional diversity among CMV promoters can be generated, and the chimeric promoters selected after two rounds of DNA shuffling and particularly designed screening assays provided approximately 2-fold increased luciferase reporter gene expression and anti-beta-galactoside antibody response in vivo when compared with wild-type promoters. Sequence analysis of the shuffled promoters identified several mutations potentially contributing to the observed enhanced or reduced promoter activities and identified a 42-nucleotide region that appears obsolete for the functioning of the CMV promoter. Taken together, these data demonstrate the feasibility of generating diverse promoter sequences by DNA shuffling and screening methods, and provide novel structure- function information about CMV promoters. DNA shuffling and screening technologies provide a new approach to promoter optimization and development of optimal expression vectors for genetic vaccinations, gene therapy, and protein expression.

Optimized expression and specific activity of IL-12 by directed molecular evolution.

DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were compared in vitro in human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration.

Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells.

CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-gamma production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.