Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited by viral vectors and its possible role in shaping these vaccine-elicited adaptive immune responses remains unclear. Here we show that Ad and Pox vectors display profound intra- and inter-group differences in innate immune cytokine and chemokine elicitation. The CD46-utilizing vectors Ad35, Ad26, and Ad48 induced greater anti-viral and proinflammatory cytokines and chemokines relative to Ad5 in vaccinated rhesus monkeys and stimulated human PBMC. Ad fiber protein, as well as other capsid components, influenced resultant Ad vector innate stimulatory phenotypes. Analysis of human sera from Ad26-vaccinated volunteers showed similar anti-viral and proinflammatory cytokine and chemokine elicitation. Mechanistic analysis of Ad innate immune stimulation showed greater amounts Ad35 and Ad26, and small amounts of Ad5, traffic to the late endosome following infection. Innate immune stimulation by all three was reduced by inhibition of endosomal acidification, Cathepsin B, and Caspase-1, suggesting a common set of innate immune sensors triggered by Ads between 0-6 hours post-infection, in agreement with trafficking data showing Ad vector colocalization in the late endosome at similar time points. These data suggest a model mechanism explaining differences in observed Ad vector innate immune stimulation phenotypes. Similar to results obtained with Ad vectors, analysis of innate cytokine and chemokine responses elicited by Pox vectors ALVAC, MVA, and NYVAC showed that all three were distinct, with the canarypox-based vector ALVAC eliciting a unique potent proinflammatory response. Together these results reveal surprising and pronounced differences in innate immune stimulatory properties of viral vectors. Furthermore, these results could lead to possible strategies for targeted construction of vaccines for desired innate immune phenotypes, and have profound implications on vaccine design against infectious diseases and cancers, as well as gene therapy.

Bovine adenovirus (BAdV)-3, a Mastadenovirus was isolated from the healthy and sick cattle (Darbyshire et al., 1965; Zhu et al., 2011). Like other adenoviruses, BAdV-3 replication is characterized by the temporally regulated expression of genes characterized by early, intermediate and late gene expression. Genus-common, non-structural protein 100K is encoded by late region L6 of BAdV-3. The objective of the present study was to characterize the BAdV-3 100K protein and identify cellular and viral proteins interacting with 100K. Although BAdV-3 100K encoded as 850 amino acid polypeptide (Reddy et al., 1998), rabbit antisera raised against peptides representing N-terminus or C-terminus recognized a protein of 130 kDa at 12-24 hrs post infection, and proteins of 130 kDa, 100 kDa, 95 kDa and 15 kDa at 36-48 hrs post infection. The 100K appeared to be localized to the nucleus and cytoplasm of BAdV-3 infected cells. In contrast, 100K localized predominantly to cytoplasm of transfected cells. However, BAdV-3 infection of cells transfected with 100K-EYFP expressing plasmid detected fluorescent protein in nucleus of the cells suggesting that another viral protein may be required for the nuclear localization of 100K. Using yeast two-hybrid and GST pull-down assays, 100K protein was shown to interact with BAdV-3 33K protein. These results were validated using bimolecular fluorescence complementation (BiFC) assay. Although, 100K protein interacts with 33K protein, co-expression of both proteins in transfected cells did not alter the cytoplasmic localization of 100K. Using GST-pull down assay and BiFC assay, 33K interacting region of 100K was localized to a stretch of 13 amino acids (624-637). Repeated attempts were not successful in rescuing a recombinant BAdV-3 expressing mutant 100K (containing deletion of amino acids 624-637). The interaction of cellular protein(s) with 100K was determined by mass spectrometric analysis of immunoprecipitated 100K. Mass spectrometry of immunoprecipitate obtained by immunoprecipitating 100K protein from BAdV-3 infected cells harvested at 48 hrs post infection identified six proteins including dynein light chain (DYNLT)1. The initial identified interaction of 100K with DYNLT1 was confirmed by the yeast two-hybrid assay, co-immunoprecipitation assay and BiFC assays. Furthermore, DYNLT1 interacting domain of 100K protein of BAdV-3 was found to be located between 499-587 amino acids. Co-expression of BAdV-3 100K-EY fusion protein with myc epitope tagged DYNLT1 protein did not alter the localization of 100K-EY fusion protein. The investigation into the differences in the subcellular localization of the 100K protein in the transfected and infected cells lead to identification of the cleavage by adenoviral protease. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (amino acid 740-745 and 781-786) in transfected or BAdV-3 infected cells. Although protease encoded by human adenovirus (HAdV)-5 or porcine adenovirus (PAdV)-3 also cleaved BAdV-3 100K at potential identified protease cleavage sites, no such cleavage of 100K encoded by HAdV-5 or PAdV-3 could be detected in cells expressing virus specific protease. Successful isolation of recombinant BAdV-3 expressing mutant protease (substitution of alanine for glycine in potential protease cleavage site) suggested that cleavage of BAdV-3 100K by viral protease is not essential for viral replication. However, further analysis observed less virus in the supernatant of cells infected with mutant BAdV-3 compared to WT BAdV-3 suggesting a possible role for cleaved C-terminal fagment in lysis of infected cells. Co-expression of BAdV-100K with other late viral proteins suggested that the 100K-EYFP fusion protein localized to the nucleus in cells co-expressing BAdV-3 protease-DsRed fusion protein. Interestingly, only C-terminal cleaved fragment of 100K localizes to the nucleus in BAdV-3 protease expressing cells. Further analysis suggested that C-terminal fragment localizing to the nucleus contains a bipartite nuclear localization signal, which is recognized by importin α3. Our results suggest that the N-terminal part of 100K may be retained in the cytoplasm by interaction with Tctex1 (DYNLT1). Our study provides for the first time a plasmid co-transfection system for the study of the protease cleavage of viral proteins. Moreover, this is the first report of cleavage of any non-structural viral proteins by adenoviral protease in infected cells.

No description available.

Necrotic enteritis (NE) is an economically important enteric disease of broiler chicken primarily caused by a-toxin (Cpa) secreted by C. perfringens type A. Mice immunized with recombinant C-terminal domain of Cpa (CpaCD) had transient and fewer localized lesions upon challenge with C. perfringens type A. These results demonstrate the usefulness of CpaCD as an immunogen for vaccine development against NE for chickens. Chicken CD40 (chCD40) is mainly expressed on the surface of chicken antigen-presenting cells (APCs), and the interaction of chCD40 and chCD40L (natural ligand for chCD40) provides crucial activation signals for chicken B-cells. A hypothesis was proposed that in ovo vaccination with an adenovirus-vectored CpaCD vaccine capable of targeting immunogen to APCs through the CD40 pathway will improve protection against NE in chickens. One agonistic monoclonal anti-chCD40 antibody (designated 2C5) was produced and characterized. 2C5 not only detected expression of chCD40 on chicken APCs, but also induced NO synthesis in chicken HD11 macrophages and enhanced proliferation of serum-starved chicken DT40 B-cells. This demonstrated substantial functional equivalence of 2C5 with chCD40L. The potential of 2C5 as an immunological adjuvant was further assessed by targeting a hapten to chicken APCs in hopes of enhancing an effective IgG response. Seven-week old chickens were immunized subcutaneously once with a complex consisting of 2C5 and peptide, and relative quantification of the peptide-specific IgG response showed that this complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. An agonistic anti-chCD40 single-chain variable fragment (designated DAG1) was combined with an adenoviral delivery system to create a vaccine, Ad-(DAG1-Cp aCD-FLAG), for in ovo administration. The efficacy of in ovo vaccination of broilers with Ad-(DAG1-Cp aCD-FLAG) in controlling NE was evaluated by C. perfringens type A challenge at 18 days post-hatch. Neither statistically significant IgA / IgG response nor protection against C. perfringens type A challenge was found in the vaccinated birds. These preliminary data suggest that a super-optimal dose of Ad-(DAG1-Cp aCD-FLAG) may be the main issue, because Cpa-specific B-cells may undergo apoptosis through the CD40 pathway.

Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication. There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not. CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59. These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC. EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber. Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC. After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells. Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.

In this study, structural and functional aspects of adenovirus type 5 (Ad5) E4orf3 protein were analyzed by biophysical and biochemical methods. Ad5 is one of the mostly used gene therapy vectors to date. However, some of its proteins possess oncogenic potential and their presence comprises safety risks. E4orf3 is one of the oncoproteins of Ad5. It also takes important roles in viral infection, and is beneficial for therapy vectors. Therefore, understanding the functions of E4orf3 is very important for developing efficient and safe adenovirus vectors. Most of the present knowledge about the functions of E4orf3 comes from its mutational analysis. It has never been expressed or purified successfully due to its extreme insolubility. Therefore, this study focused on the optimization of expression of E4orf3 protein. As a result, full-length E4orf3 was obtained in soluble form as a Glutathione-S-transferase (GST) fusion protein and purified by GST affinity chromatography for the first time. Subsequently, the interaction of E4orf3 with four different proteins, DNA-PK, Aup1, E1B-55 kDa and E4orf6 was analyzed in detail by GST-pulldown technique. In these experiments, E4orf3 was shown to associate with Aup1, E1B-55 kDa and E4orf6 in vitro, and the C-terminal of E4orf3 was determined to be responsible for these interactions. Finally, basic structural information about E4orf3 protein was also obtained for the first time by the direct analysis of the fusion protein in glutathione beads with Fourier Transform Infrared (FTIR) spectroscopy. Since the purified E4orf3 protein could not be separated from the glutathione beads due to its hydrophobic regions, the secondary structures in this protein were determined after subtracting glutathione and H2O absorption bands, and the GST moiety.

En aquest estudi es mostra clarament que en hepatòcits la Glu 6-P produïda per la HK I no pot ser dirigida cap al glicogen ja que no pot activar la GS de fetge. Aquesta evidència sembla indicar que la GK és l'element que facilita que l'hexosa fosforilada activi la LGS i serveixi per a la síntesi de glicogen en hepatòcits. A més, l'activació de la GS per part de la Glu 6-P de la gluconeogènesi, tal i com s'ha demostrat en els estudis presentats, reforça la idea que la GK no és la única, ni imprescindible per a la funcionalitat de la GS hepàtica, sinó que la HK I n'és l'excepció.D'aquests estudis, també podem concloure que l'acumulació hepàtica de glicogen a partir de glucosa està sotmesa a un sistema de control en el qual el controlador, GS, és alhora controlat per la GK. Aquest sistema és diferent de les cascades de fosforilació, en el qual l'enzim controla directament l'activitat del que el succeeix en la sèrie. En el nostre sistema, el control de manera indirecta l'exerceixen els nivells de Glu 6-P, que engeguen la desfosforilació de la GS, i per tant l'activen. Per això considerem la Glu 6-P tant un precursor com una molècula de senyalització que dirigeix la incorporació de glucosa a glicogenPer aprofundir en l'estudi del mecanisme d'activació de la LGS per glucosa s'han utilitzat les cèl·lules FTO-2B aprofitant que no expressen la GK. Fins aquest moment, s'acceptava que la glucosa quan era fosforilada per la GK estimulava l'activació de la GS millor que si era fosforilada per la HK I. La LGS endògena s'activa en resposta a concentracions creixents de glucosa quan la GK hi ha estat expressada però no quan la font de Glu 6-P és la HK I endògena o HK I sobreexpressada. Això suggereix la presència de com a mínim dues poblacions de Glu 6-P en la cèl·lula. Una d'aquestes poblacions prové de l'acció de la GK i és accessible a la LGS, mentre que la població de molècules de Glu 6-P produïdes per la HK I es localitzen en un compartiment cel·lular del qual està exclosa la LGS. En aquestes s'ha observat que la LGS, però no la MGS, diferència entre la Glu 6-P produïda per la GK o per la HK I. La isoforma hepàtica presenta especificitat per l'origen d'aquest metabòlit a diferència de la isoforma muscular, cosa que reforça les diferencies en els mecanismes de la síntesi del glicogen en el fetge i el múscul.Tots aquests resultats i aquells descrits a la bibliografia amb anterioritat, ens permeten concloure: primer, que la síntesi de glicogen succeeix a la perifèria de l'hepatòcit, i és funció de l'activació de la GS via Glu 6-P. En la perifèria cel·lular és on, probablement, es donen les condicions òptimes, com l'accés a substrats, per a la síntesi de glicogen.Segon, que la font utilitzada per sintetitzar la Glu 6-P es determinant per activar la GS. En el cas que s'utilitzi un precursor gluconeogènic per sintetitzar glicogen, la G6Pasa juga un paper clau al decidir en quina direcció (producció de glucosa o síntesi de glicogen) s'utilitzarà la Glu 6-P. En aquest sentit, recentment s'ha descrit que in vivo la inhibició de la Glu 6-P translocasa porta a una utilització de la Glu 6-P produïda per gluconeogènesi cap a la síntesi de glicogen. Tercer, quan la Glu 6-P prové de la fosforilació de glucosa per part de la GK, la síntesi de glicogen incrementa en funció dels nivells de glucosa. El glicogen es produeix de manera més eficient gràcies a la inactivació de la GP per glucosa i la conseqüent disminució de la degradació del glicogen. El funcionament tan acurat d'aquesta maquinària esdevé essencial en el balanç entre la producció hepàtica de glucosa i la síntesi de glicogen.

Regensburg, Univ., Diss., 2008.