Natl. the coxsackievirus and adenovirus receptor (CAR). These differences in innate triggering result in markedly different immunologic milieus for the subsequent generation of adaptive immune responses by these vaccine vectors. INTRODUCTION Adenovirus (Ad) vectors are widely used for vaccination due to their immunogenicity, relatively large transgene coding capacity, and multiple available serotypes with diverse biological properties. While considerable data have been generated regarding adaptive immune responses elicited by Ad vectors, much less is known about CDK8-IN-1 innate immune responses CDK8-IN-1 induced by these vectors. As innate immune induction is critical for understanding both reactogenicity and adaptive immunity, it is important to define the innate pathways triggered by Ad vectors from various serotypes. Accumulating evidence suggests that different serotype Ad vectors induce qualitatively different adaptive immune response phenotypes (1, 23, 27, 42). In particular, vaccine studies using the simian immunodeficiency virus (SIV) infection model in rhesus monkeys have shown qualitative differences in adaptive immune responses elicited by various serotype Ad vectors (27) which translated into different levels of protective efficacy against SIV challenges (6, 7, 28). However, innate immune profiles of different alternative serotype Ad vectors have not previously been studied in nonhuman primates. Adenoviruses are a diverse group of double-stranded DNA viruses with at least 65 known human serotypes, which are subdivided into species A to G based upon sequence homology (10, 26, 30, 49). Vectors constructed using these viruses have been shown to differ significantly in terms of primary receptor usage (1, 9, 13, 40, 50), intracellular trafficking patterns (14, 22, 31, 32), transduction and activation of dendritic cells (2, 11, 20, 29, 36, 53), utilization of secondary receptors (15, 48), cellular tropism (3, 16, 33, 44, 46, 47), and interaction with pattern recognition receptors (PRR) (12, 18, 35). The species C adenovirus serotype 5 (Ad5), the species B2 adenovirus serotype 35 (Ad35), and the species D adenovirus serotype 26 (Ad26) are currently being evaluated as vaccine candidates in clinical trials, yet relatively little is known about the possible differences in innate immunity induced by these vectors. Notably, Ad5 utilizes the coxsackievirus and adenovirus receptor (CAR) as its primary cellular receptor, whereas Ad35, Ad26, and Ad48 utilize CD46 (24). In this study, Zfp622 we describe the innate cytokine profiles induced by Ad vectors from 5 serotypes in 26 rhesus monkeys. We then assessed the mechanism of differential viral triggering of these innate responses using capsid chimeric vectors and receptor-blocking monoclonal antibodies (MAbs) in human peripheral blood mononuclear cells (PBMC). Our studies demonstrate that Ad35, Ad26, and Ad48 vectors that utilize CD46 as their primary cellular receptor trigger innate cytokine profiles characterized by higher levels of antiviral and proinflammatory cytokines and chemokines than those triggered by Ad5 vectors that utilize CAR. MATERIALS AND METHODS Viruses. E1/E3-deleted vectors Ad5, Ad35, Ad26, Ad48, and chimeric Ad5 with the hexon hypervariable regions (HVRs) replaced with those of Ad48 (Ad5HVR48) expressing SIV antigens and no transgene were produced as previously described (1, 34). Briefly, vectors were produced by recombination in E1-complementing PER.55K cells and were purified by CsCl density centrifugation. Cells. Normal human blood was collected in the presence of sodium heparin and processed by the Ficoll-Hypaque gradient method as previously described (8). Cells were resuspended in R10 medium (RPMI, 10% fetal calf serum [FCS], 50 U/ml penicillin, 50 g/ml streptomycin) at a concentration of 1 1 106 cells/ml and utilized in assays. All studies involving human subjects were approved by the Beth Israel Deaconess Medical Center Institutional Review Board (IRB). Antibodies. The anti-CAR MAb RmcB (Millipore, Billerica, MA) and anti-CD46 MAbs 13/42 (LifeSpan Biosciences, Seattle, WA) and CDK8-IN-1 M177 (Hycult Biotechnology, Plymouth Meeting, PA), as well as the anti-KLH mouse IgG isotype control (R&D Systems, Minneapolis, MN), were washed 3 with 1 ml unsupplemented Dulbecco’s phosphate-buffered saline (DPBS) and concentrated to 1 1 g/l by centrifugation at 3,000 rpm in Amicon Ultra-4 centrifugal filters (molecular mass, 30 kDa) (Millipore, Billerica, MA) and stored at 4C for immediate use. Flow cytometry antibody panels included CD3-allophycocyanin (UCHT1), CD16-fluorscein isothiocyanate (3G8), CD123-peridinin chlorophyll protein-Cy5.5 (9F5), CD11c-phycoerythrin (B-ly6), CD56-phycoerythrin-Cy7 (B159), CD19-V450 (HlB19), CD14-allophycocyanin-Cy7 (MP9) (BD Biosciences, San Diego, CA), and HLA-DR-AlexaFluor700 (LN3) (eBioscience, San Diego, CA). cytokine stimulation assay. A total of 1 1 106 PBMC were incubated with 1,000 viral particles (vp)/cell of the indicated adenovirus vector. In certain experiments, cells were preincubated with MAbs for 1 h prior to infection. Cells were cultured at 37C, and supernatants were.
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