Pre-Existing Adenovirus Immunity Modifies a Complex Mixed Th1 and Th2 Cytokine Response to an Ad5/HIV-1 Vaccine Candidate in Humans

The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest that vector-specific humoral responses may reduce vaccine-induced T-cell responses by previously undetected mechanisms

NLRP3 Inflammasome: Key Mediator of Neuroinflammation in Murine Japanese Encephalitis

Background: Japanese Encephalitis virus (JEV) is a common cause of acute and epidemic viral encephalitis. JEV infection is associated with microglial activation resulting in the production of pro-inflammatory cytokines including Interleukin-1 b (IL-1b) and Interleukin-18 (IL-18). The Pattern Recognition Receptors (PRRs) and the underlying mechanism by which microglia identify the viral particle leading to the production of these cytokines is unknown. Methodology/Principal Findings: For our studies, we have used murine model of JEV infection as well as BV-2 mouse microglia cell line. In this study, we have identified a signalling pathway which leads to the activation of caspase-1 as the key enzyme responsible for the maturation of both IL-1b and IL-18 in NACHT, LRR and PYD domains-containing protein-3 (NLRP3) dependent manner. Depletion of NLRP3 results in the reduction of caspase-1 activity and subsequent production of these cytokines. Conclusion/Significance: Our results identify a mechanism mediated by Reactive Oxygen Species (ROS) production and potassium efflux as the two danger signals that link JEV infection to caspase-1 activation resulting in subsequent IL-1b an

Gene Transfer of Mutant Mouse Cholinesterase Provides High Lifetime Expression and Reduced Cocaine Responses with No Evident Toxicity

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with (3)H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3×10(11) particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (10(10) particles) to 20,000 fold (10(13) particles), while the hdAD vector (1.7×10(12) particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7×10(12) particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose

Microparticles and their impact on intestinal immunity

Microparticles are small (<1 µm), nonbiological particles that are used in many areas of daily life. As food additive they are used as anticaking agents or food colorants. The most common food-derived ingested compounds are aluminium silicate and titanium dioxide (TiO(2)), the latter being a white pigment used in toothpaste or sugar toppings. The increasing abundance of microparticles in the Western diet raises the question of the potential risks associated with gastrointestinal diseases such as Crohn's disease (CD). Accumulation of particles has been shown in cells of Peyer's patches, but it is not clear whether this also has pathological effects. NLRP3 is a member of the intracellular pattern recognition receptor family and it is part of the inflammasome, a multiprotein complex containing caspase-1 which activates the proinflammatory cytokines interleukin (IL)-1β and IL-18. With regard to recent findings identifying small particles such as asbestos and monosodium urate as NLRP3 activators, TiO(2) may be another potential target for inflammasome studies. We found that macrophage-like cells readily take up TiO(2) after 6 h. Incubation of cells with TiO(2) resulted in the assembly of NLRP3 with caspase-1. This inflammasome assembly correlated with secretion of IL-1β. In intestinal epithelial cells, TiO(2) also was found to be ingested. The counting of particles localized intracellularly revealed a dose-dependent increase of TiO(2)-positive cells. This points to the fact that in humans with a leaky intestinal barrier (such as IBD patients), TiO(2) microparticles may be taken up by macrophages and intestinal epithelial cells, may activate the inflammasome and induce IL-1β and IL-18 secretion. This may aggravate inflammation in susceptible individuals