Adenovirus and adeno-associated virus vectors

Recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) are among the most extensively used vectors in gene therapy studies to date. These two vectors share some similar features such as a broad host range and ability to infect both proliferating and quiescent cells. However, they also possess their own unique set of properties that render them particularly attractive for gene therapy applications. rAd vectors can accommodate larger inserts, mediate transient but high levels of protein expression, and can be easily produced at high titers. Development of gutted rAd vectors has further increased the cloning capacity of these vectors. The gaining popularity of rAAV use in gene therapy can be attributed to its lack of pathogenicity and added safety due to its replication defectiveness, and its ability to mediate long-term expression in a variety of tissues. Site-specific integration, as occurs with wild-type AAV, will be a unique and valuable feature if incorporated into rAAV vectors, further improving their safety. This paper describes these properties of rAd and rAAV vectors, and discusses further development and vector improvements that continue to extend the utility of these vectors, such as cell retargeting by capsid modification, differential transduction by use of serotypes, and extension of the cloning capacity of rAAV vectors by dual vector heterodimerization.     

Transduction profiles of recombinant adeno-associated virus vectors derived from serotypes 2 and 5 in the nigrostriatal system of rats

We compared the transduction efficiencies and tropisms of titer-matched recombinant adeno-associated viruses (rAAV) derived from serotypes 2 and 5 (rAAV-2 and rAAV-5, respectively) within the rat nigrostriatal system. The two serotypes (expressing enhanced green fluorescent protein [EGFP]) were delivered by stereotaxic surgery into the same animals but different hemispheres of the striatum (STR), the substantia nigra (SN), or the medial forebrain bundle (MFB). While both serotypes transduced neurons effectively within the STR, rAAV-5 resulted in a much larger EGFP-expressing area than did rAAV-2. However, neurons transduced with rAAV-2 vectors expressed higher levels of EGFP. Consistent with this result, EGFP-positive projections emanating from transduced striatal neurons covered a larger area of the SN pars reticulata (SNr) after striatal delivery of rAAV-5, but EGFP levels in fibers of the SNr were higher after striatal injection of rAAV-2. We also compared the potentials of the two vectors for retrograde transduction and found that striatal delivery of rAAV-5 resulted in significantly more transduced dopaminergic cell bodies within the SN pars compacta and ventral tegmental area. Similarly, EGFP-transduced striatal neurons were detected only after nigral delivery of rAAV-5. Furthermore, we demonstrate that after striatal AAV-5 vector delivery, the transduction profiles were stable for as long as 9 months. Finally, although we did not target the hippocampus directly, efficient and widespread transduction of hippocampal neurons was observed after delivery of rAAV-5, but not rAAV-2, into the MFB.     

Abeta42 mutants with different aggregation profiles induce distinct pathologies in Drosophila

Aggregation of the amyloid-beta-42 (Abeta42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Abeta aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Abeta can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Abeta42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Abeta42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Abeta42Arc) and an artificial mutation (Abeta42art) that is known to suppress aggregation and toxicity of Abeta42 in vitro. In the Drosophila brain, Abeta42Arc formed more oligomers and deposits than did wild type Abeta42, while Abeta42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Abeta peptides. Surprisingly, however, Abeta42art caused earlier onset of memory defects than Abeta42. More remarkably, each Abeta induced qualitatively different pathologies. Abeta42Arc caused greater neuron loss than did Abeta42, while Abeta42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Abeta aggregates: Abeta42Arc formed large deposits in the cell body, Abeta42art accumulated preferentially in the neurites, while Abeta42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Abeta42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.     

Sperm cells induce distinct cytokine response in peripheral mononuclear cells from infertile women with serum anti-sperm antibodies

Background and Aims

Anti-sperm antibodies in can markedly reduce the likelihood of natural conception. The etiology of this anti-sperm immunity in human females is unknown. We compared the cytokine response of peripheral blood mononuclear cells (PBMCs) from infertile patients with or without anti-sperm antibodies (ASA) and fertile women.

Methodology/Principal Findings

We cultivated the PBMCs together with sperm antigens (whole cells or cell lysate), and screened the supernatants for 40 cytokines by antibody array. When stimulated with whole sperm cells, the PBMCs from patients with ASA produce less IL-3, IL-11, IL-13, ICAM-1, GCSF and more IL-2, IL-4 and IL-12p70 as compared to healthy women. PBMCs from patients with ASA produce typically less IL-13, IL-7, IL-17 and MIG, and more MIP-1β and IL-8, as compared to PBMCs from patients without ASA. In response to sperm cell lysate, PBMCs from infertile women without ASA respond initially by increase in production of growth factors (GCSF, GM-CSF and PDGF-BB) followed by increase in chemokines (e.g. IL-8, MCP-1 and MIP-1β).

Conclusions

Cellular immune responses to sperm antigens, measured by production of cytokines, differ among infertile women with ASA, infertile women without ASA and healthy women. This difference could play an important role in the initial steps of the infertility pathogenesis.     

Transient acidification and subsequent proinflammatory cytokine stimulation of astrocytes induce distinct activation phenotypes

The foot processes of astrocytes cover over 60% of the surface of brain microvascular endothelial cells, regulating tight junction integrity. Retraction of astrocyte foot processes has been postulated to be a key mechanism in pathology. Therefore, movement of an astrocyte in response to a proinflammatory cytokine or even limited retraction of processes would result in leaky junctions between endothelial cells. Astrocytes lie at the gateway to the CNS and are instrumental in controlling leukocyte entry. Cultured astrocytes typically have a polygonal morphology until stimulated. We hypothesized that cultured astrocytes which were induced to stellate would have an activated phenotype compared with polygonal cells. We investigated the activation of astrocytes derived from adult macaques to the cytokine TNF‐α under resting and stellated conditions by four parameters: morphology, intermediate filament expression, adhesion, and cytokine secretion. Astrocytes were stellated following transient acidification; resulting in increased expression of GFAP and vimentin. Stellation was accompanied by decreased adhesion that could be recovered with proinflammatory cytokine treatment. Surprisingly, there was decreased secretion of proinflammatory cytokines by stellated astrocytes compared with polygonal cells. These results suggest that astrocytes are capable of multiple phenotypes depending on the stimulus and the order stimuli are applied. J. Cell. Physiol. 228: 1284–1294, 2013. © 2012 Wiley Periodicals, Inc.     

Adenovirus vectors for gene expression.

Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates.     

Adenovirus vectors for gene delivery.

Recent endeavors in the development of adenovirus as a gene vector have focused on the modification of virus tropism, the accommodation of larger genes, and the increase in stability and control of transgene expression. Whereas partial or total deletions of viral genes increase the cloning capacity and partly reduce the cellular immune response, control of the humoral response, which often precludes efficient readministration, remains a challenge.     

Adenovirus vectors activate survival pathways in lung epithelial cells

Airway epithelial cells are often the sites of targeted adenovirus vector delivery. Activation of the host inflammatory response and modulation of signal transduction pathways by adenovirus vectors have been previously documented, including activation of MAP kinases and phosphatidylinositol 3-kinase (PI3-kinase). The effect of activation of these pathways by adenovirus vectors on cell survival has not been examined. Both the PI3-kinase/Akt and ERK/MAP kinase signaling pathways have been linked to cell survival. Akt has been found to play a role in cell survival and apoptosis through its downstream effects on apoptosis-related proteins. Constitutive activation of either PI3-kinase or Akt blocks apoptosis induced by c-Myc, UV radiation, transforming growth factor-beta, Fas, and respiratory syncytial virus infection. We examined the effect of adenovirus vector infection on activation of these prosurvival pathways and its downstream consequences. Airway epithelial cells were transduced with replication-deficient adenoviral vectors containing a nonspecific transgene, green fluorescent protein driven by the cytomegalovirus promoter, or an empty vector with no transgene. They were then exposed to the proapoptotic stimulus actinomycin D plus TNF-alpha, and evidence of apoptosis was evaluated. Compared with the cells treated with actinomycin/TNF alone, the adenovirus vector-infected cells had a 50% reduction in apoptosis. When we examined induction of the prosurvival pathways, ERK and AKT, in the viral vector-infected cells, we found that there was significant activation of both Akt and ERK.     

Pneumococcal serotypes in various diseases of children

The serotyping of pneumococci isolated from different material obtained from children aged 0 to 11 years was carried out. Out of 156 patients with different diseases, hospitalized in two clinics in Moscow during February-May 1983, pneumococci were isolated from 67 patients (43%). The isolated pneumococcal strains belonged to 11 serotypes. Pneumococci of serotypes 3, 6, 9 and 19 were shown to occur most frequently in different diseases and constituted 50% of the isolated strains. The inoculation of the material by the quantitative method permitted the authors to find out the role of pneumococci as the etiological factor in the pathogenesis of some diseases. A certain dependence of diversity in the types of isolated pneumococci on the age of sick children was noted. Almost all isolated strains were found to be sensitive to penicillin, ampicillin and benzylpenicillin. But a few individual strains were sensitive only to one of these antibiotics. The data on some biological properties of pneumococci cultivated on solid culture media are presented.     

Macrophage-activating factors from different T cell clones induce distinct macrophage functions

The data reported in this paper are the first demonstration that different T cell clones (PC-AKR-CI 96, clone 96; PK 7.1.2 E8, clone 7.1.2 E8) secrete different macrophage-activating factors (MAF) that induce distinct macrophage activities. Incubation of resident murine macrophages with MAF 7.1.2 E8 increased RNA, protein, and glycoprotein synthesis, hexosemonophosphate shunt (HMPS) activity, release of oxygen metabolites (O-2, H2O2), pinocytosis, phagocytosis, and tumor cytostasis, whereas no effect on prostaglandin E (PGE) release, schistosomula killing, and tumor cytolysis could be observed. In contrast, MAF 96 increased glycoprotein synthesis, HMPS activity, release of oxygen metabolites and PGE, schistosomula killing, and tumor cytostasis and cytolysis, whereas RNA and protein synthesis and pinocytosis were decreased and phagocytosis remained unaffected. Thus, MAF from both T cell clones share some macrophage-activating properties but differ in others. Most importantly, both MAF could be differentiated serologically by a rabbit anti-lymphokine antiserum that selectively inhibited MAF 96 but not MAF 7.1.2 E8 activity. At optimal concentrations, MAF 96 and 7.1.2 E8 were active in the absence of lipopolysaccharide (LPS) whereas LPS enhanced the activity of suboptimal doses of MAF 96 but not of MAF 7.1.2 E8. These data are discussed with respect to the possibility that the functional dichotomy of T cell clones might reflect different activities of normal T cell subpopulations.     

Different respiratory syncytial virus and Quillaja saponin formulations induce murine peritoneal cells to express different proinflammatory cytokine profiles

The recognition of a pathogen or a vaccine antigen formulation by cells in the innate immune system leads to production of proinflammatory cytokines, which will determine the ensuing acquired immune response quantitatively and qualitatively. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 are the first set of cytokines produced upon such an encounter, which have roles both in protective immunity and immunopathogenesis evident with respiratory syncytial virus (RSV). RSV antigens in different physical adjuvant-vaccine formulations were analysed for their capacity to provoke cultured murine peritoneal cells to produce these three proinflammatory cytokines. RSV immunostimulating complex (ISCOM), i.e. both antigen and adjuvant are incorporated in the same particle, induced high levels of IL-1alpha being of the same magnitude or higher than those of live RSV and lipopolysaccharide (LPS). Live virus and LPS induced higher levels of IL-6 and TNF-alpha than ISCOM and so did non-adjuvanted UV-inactivated RSV but only at high doses. ISCOM-Matrix, i.e. ISCOM without antigens, admixed as a separate entity to inactivated RSV, downregulated or blocked the cytokine response to the inactivated RSV in contrast to ISCOM. Kinetic studies showed that ISCOM induced cytokine production first detected at hours 1, 2, 4 for TNF-alpha, IL-6 and IL-1alpha respectively, which was earlier than for the other antigen formulations containing corresponding doses of antigen and/or Quillaja adjuvant. Peak values for production of TNF-alpha and IL-6 were at 8 h and for IL-1alpha at 72 h following stimulation with ISCOM. The delayed appearance of IL-1alpha may reflect the cell-bound nature of this cytokine.     

Electron microscopic examination of capsular material from various serotypes of Actinobacillus pleuropneumoniae.

The capsular material on PPLO broth-grown cells of Actinobacillus pleuropneumoniae representing serotypes 1 to 10 was visualized by transmission electron microscopy after polycationic ferritin labeling and also after stabilization with specific antibodies. All the isolates examined were covered with a layer of capsular material whose thickness varied between 80 to 90 nm and 210 to 230 nm when examined by immunostabilization. We were also able to visualize A. pleuropneumoniae in lungs of infected pigs and to estimate the amount of capsular material covering the cells. Our results indicate that differences in capsular structure exist among the different A. pleuropneumoniae serotypes, and this result may explain in part why the serotypes are not equally virulent.     

Production methods for gene transfer vectors based on adeno-associated virus serotypes

Vectors derived from adeno-associated virus serotype 2 (AAV-2) represent a most promising tool for human gene transfer because these vectors are neither pathogenic nor toxic to the target cell, and allow long-term gene expression in a large variety of tissues. However, they are rather inefficient at infecting a number of clinically relevant cell types, and transduction by these vectors is likely hampered by neutralizing antibodies that are highly prevalent in the human population. Therefore, an increasing number of researchers are currently turning their attention to the five other serotypes of AAV, to try and develop these as novel vectors for human gene transfer, hoping to overcome the problems associated with AAV-2 vectors. Here I describe and discuss the methodology to produce these alternative AAV vectors in tissue culture. In detail, two strategies are compared that rely on transfection of cells in culture with either two or three plasmids, containing the AAV vector genome and encoding AAV and adenoviral helper functions. Either of these protocols can be used to package a recombinant AAV genome into capsids of its own serotype (generation of "real" serotypes) or to "cross-package" this vector DNA into capsids derived from another AAV serotype ("pseudotyping"). As these approaches are still in their early stages, the existing limitations of current technology are discussed, and possible further improvements proposed.     

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.