Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method.

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.

ß-Carotene Biosynthesis in Probiotic Bacteria.

Susceptibility to deadly diarrheal diseases is partly due to widespread pediatric vitamin A deficiency. To increase vitamin A coverage in malnourished children, we propose to engineer a probiotic bacterium that will produce ß-carotene in the intestine, which will be metabolized to vitamin A. Such a therapy has the potential to broadly stimulate mucosal immunity and simultaneously reduce the incidence and duration of diarrheal disease. To that end, a ß-carotene-producing variant of the probiotic Escherichia coli strain Nissle 1917 (EcN-BETA) was generated. Notably, the strain produces ß-carotene under anaerobic conditions, reflective of the gut environment. EcN-BETA also retains ß-carotene production capability after lyophilization, suggesting that it may be amenable to dry formulation. Moreover, EcN-BETA activates murine dendritic cells in vitro, suggesting that the presence of ß-carotene may not diminish the immunostimulatory capacity of EcN. Finally, we present a framework through which further improvements may enable approaches such as the one described in this report to yield innovative life-saving therapies for the developing world.

Nociceptin/orphanin FQ inhibition with SB612111 ameliorates dextran sodium sulfate-induced colitis.

Inflammatory bowel diseases, primarily Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract with unknown etiology. The majority of current therapeutic agents focus on controlling proinflammatory molecules. The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been described as a potential immunomodulator for inflammatory bowel diseases. In this study, we asked whether the small molecule N/OFQ antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB612111) would inhibit the development of dextran sodium sulfate-induced colitis in C57BL/6 mice. Inhibition of the N/OFQ receptor (NOP) by SB612111 significantly ameliorated the clinical disease course in these animals, as indicated by reduced fecal bleeding, improved recovery from diarrhea and weight loss, and a reduction in histopathological alterations. In addition, the inflammatory response in the colon was diminished, as demonstrated by reduced cytokine protein and messenger RNA expression for CXCL1/keratinocyte-derived chemokine, interferon-γ, interleukin-1ß, interleukin-6, and tumor necrosis factor-α, some of which are known targets for the treatment of this devastating disease. Our results strongly support a role for the receptor-ligand pair NOP-N/OFQ in the pathogenesis of colitis. We conclude that inhibition of NOP receptors with small molecule inhibitors may constitute a novel, urgently needed approach for the treatment of inflammatory bowel diseases.

Immunotoxicity of gallium arsenide on antigen presentation: comparative study of intratracheal and intraperitoneal exposure routes.

Gallium arsenide (GaAs) is a semiconductor utilized in electronics and computer industries. GaAs exposure of animals causes local inflammation and systemic immune suppression. Mice were administered 2 to 200 mg/kg GaAs. On day 5, intratracheal instillation increased lung weights in a dose-dependent manner and induced pulmonary inflammation exemplified by mononuclear cell infiltration and mild epithelial hyperplasia. No fibrosis, pneumocyte hyperplasia, proteinosis, or bronchial epithelial damage was observed in the lungs. Splenic cellularity and composition were unaffected. GaAs' effect on antigen presentation by macrophages was similar after intratracheal and intraperitoneal exposure, although the lowest observable adverse effect levels differed. Macrophages from the exposure site displayed an enhanced ability to activate an antigen-specific CD4(+) helper T-cell hybridoma compared with vehicle controls, whereas splenic macrophages were defective in this function. The chemical's impact on peritoneal macrophages depended on the exposure route. GaAs exposure augmented thiol cathepsins B and L activities in macrophages from the exposure site, but decreased proteolytic activities in splenic macrophages. Alveolar macrophages had increased expression of major histocompatibility complex (MHC) Class II molecules, whereas MHC Class II expression on splenic and peritoneal macrophages was unaffected. Modified thiol cathepsin activities statistically correlated with altered efficiency of antigen presentation, whereas MHC Class II expression did not. Our study is the first one to examine the functional capability of alveolar macrophages after intratracheal GaAs instillation. Therefore, thiol cathepsins may be potential target molecules by which GaAs exposure modulates antigen presentation.

Impact of in vitro gallium arsenide exposure on macrophages.

The semiconductor gallium arsenide (GaAs) is classified as an immunotoxicant and a carcinogen. We previously showed that GaAs in vivo induces several phenotypic changes in macrophages located at the exposure site, indicative of an activated state. These physiological alterations may be a primary or secondary consequence of chemical exposure. To discern primary influences, our current study examined the in vitro effects of the chemical on macrophage cell lines and murine peritoneal macrophages. GaAs augmented cathepsins L and B proteolytic activities in all three sources of macrophages. Expression of the two mature isoforms of invariant chain and its cleavage fragment was also significantly increased, indicating that the chemical directly affects macrophages. However, GaAs did not alter the overall cell surface expression of major histocompatibility complex class II molecules on macrophages nor influence their ability to stimulate antigen-specific helper T cell hybridomas to respond to intact antigens that require processing. These findings raise the possibility that the chemical's complete in vivo impact may involve cytokines. Further, GaAs in vitro enhanced steady-state cathepsin L protein, and cathepsins L and B mRNA expression in macrophages, indicating that GaAs may alter gene expression, which may contribute to the chemical's adverse biological effects.