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Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy. [ABSTRACT FROM AUTHOR]

Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy. [ABSTRACT FROM AUTHOR]

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-κB, a positive regulator of IL-8 expression, and decreased expression of IkB-α, the inhibitory protein of NF-κB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is colocalized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1. [ABSTRACT FROM AUTHOR]

Introduction: Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation.Methods: Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry.Results: Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit.Conclusions: Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation.Trial Registration Number: NCT00974064; NCT01776398. [ABSTRACT FROM AUTHOR]

An abstract of the article "Antigen Epitopes Expressed in Different Adenovirus Capsid Proteins Induce Different Levels of Epitope-Specific Immunity," by Anja Krause and colleagues is presented.

An abstract of the article "Immunization with an Adenovirus Vector Expressing a P. aeruginosa OprF Epitope in the Capsid Enables Boosting of Anti-Epitope Immune Responses and Protects Against Pulmonary Infection with P. aeruginosa," by Stefan Worgall and colleagues is presented.

Respiratory syncytial virus (RSV) is the leading cause of severe viral respiratory disease in infants and children. The morbidity is greatest in young infants, children with underlying risk factors such as prematurity and bronchopulmonary dysplasia, and the elderly. A safe and efficient active vaccine is not available. Passive immunization with the monoclonal antibody palivizumab, a humanized murine IgG1 antibody targeted against a neutralizing epitope of the F glycoprotein of RSV, diminishes disease severity in high risk populations and is routinely used to protect premature infants during the RSV season. Because palivzumab offers only short-term protection and must be administered every 30 days, we hypothesized that genetic delivery of palivizumab with gene therapy vectors could be a means to achieve long term protection against RSV infection. To test the hypothesis, we designed a murine version of the palivizumab antibody by replacing the human sequences with the sequence from the constant region of a murine antibody, while preserving the complementarity-determining region. As a proof-of- principle to test the validity of the strategy, an adenovirus (Ad) vector was used, and to achieve long term protection, an adenoassociate virus (AAV) was used. Using an Ad vector expressing genes for the light and heavy chains of the modified palivizumab antibody separated by an internal ribosome entry site (Adanti-RSV) was constructed. The light and heavy chains were detected by Western analysis in the supernatant of 293 cells infected with Adanti- RSV, but not in cells infected with the control Ad vector AdNull, and the antibody secreted by the 293 cells bound RSV as analyzed by SDS gel electrophoresis. To determine if Adanti-RSV would mediate generation of anti-RSV antibodies in vivo, 5x1010 particle units of Adanti-RSV or AdNull., was administered to BALB/c mice intravenously. After 4 days, RSV neutralizing titers (1:160) were detected in mice receiving Adanti-RSV but not in AdNull infected or naive mice. Four days following vector administration, the mice were challenged by intranasal administration of RSV virus strain A2 (106 pfu) and the lung homogenates analyzed for RSV titers 4 days later. The mice that had received Adanti-RSV had 2.5-fold lower titers of RSV in the lung following challenge compared to the AdNull or naive group (p<0.05). To evaluate long term protection, the anti- RSV antibody construct was expressed in an serotype rh10 AAV vector. Six weeks following intrapleural administration 5x1010 genome copies, of AAVrh10.anti-RSV or the control AAVrh10.α1AT to BALB/c mice, the mice were challenged with RSV. Mice that had received AAVrh10. anti-RSV showed lower 2.5-fold RSV titers in the lung compared to the AAVrh10.α1AT group (p<0.05). Together these data demonstrate that a gene transfer strategy for delivery of an anti-RSV antibody can generate protective immunity in mice against RSV infection to the respiratory tract and may provide an alternative to the administration of the antibody itself.Molecular Therapy (2006) 13, S301–S302; doi: 10.1016/j.ymthe.2006.08.866 [ABSTRACT FROM AUTHOR]

An abstract of the article "Protective Immunity Against Yersinia pestis in Mice Immunized with an Ad-Based Vaccine Vector Expressing the Y. pestis F1 and V Antigens," by Maria Chiuchiolo, Julie L. Boyer, Tiffany Niven, Anja Krause, Neil R. Hackett and Ronald G. Crystal is presented.

Autophagy is an intracellular pathway that contributes to the degradation and recycling of unfolded proteins. Based on the knowledge that autophagy affects glycogen metabolism and that alpha(1)-antitrypsin (AAT) deficiency is associated with an autophagic response in the liver, we hypothesized that the conformational abnormalities of the Z-AAT protein interfere with hepatocyte glycogen storage and/or metabolism. Compared with wild-type mice (WT), the Z-AAT mice had lower liver glycogen stores (P < 0.001) and abnormal activities of glycogen-related enzymes, including acid alpha-glucosidase (P < 0.05) and the total glycogen synthase (P < 0.05). As metabolic consequences, PiZ mice demonstrated lower blood glucose levels (P < 0.05), lower body weights (P < 0.001), and lower fat pad weights (P < 0.001) compared with WT. After the stress of fasting or partial hepatectomy, PiZ mice had further reduced liver glycogen and lower blood glucose levels (both P < 0.05 compared WT). Finally, PiZ mice exhibited decreased survival after partial hepatectomy (P < 0.01 compared with WT), but this was normalized with postoperative dextrose supplementation. In conclusion, these observations are consistent with the general concept that abnormal protein conformation and degradation affects other cellular functions, suggesting that diseases in the liver might benefit from metabolic compensation if glycogen metabolism is affected.