A porcine adenovirus with low human seroprevalence is a promising alternative vaccine vector to human adenovirus 5 in an H5N1 virus disease model

Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5.     

Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA.     

Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.     

A comparative analysis of numt evolution in human and chimpanzee

Mitochondrial DNA sequences are frequently transferred into the nuclear genome, giving rise to numts (nuclear DNA sequences of mitochondrial origin). So far, the evolutionary history of numts has largely been studied by using single genomes. Here, we present the first attempt to study numt evolution in a comparative manner by using a pairwise genomic alignment. The total number of numts was estimated to be 452 in human and 469 in chimpanzee. numts that were found in both genomes at identical loci were deemed to be orthologous; 391 numts (>80%) were classified as such. The preponderance of orthologous numts is due to the very short divergence time between the 2 hominoids. The rest of numts were deemed to be nonorthologous. Nonorthologous numts were subdivided into 1) ancestral numts that have lost an ortholog in one species through deletion (12 in human and 11 in chimpanzee), 2) new numts acquired by the insertion of a mitochondrial sequence after the divergence of the 2 species (34 in human and 46 in chimpanzee), and 3) paralogous numts created by the tandem duplication of a preexisting numt (2 in human). This approach also enabled us to reconstruct the numt repertoire in the common ancestor of humans and chimpanzees (409 numts). Our comparative approach is also useful in identifying the exact boundaries of numts.     

A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.     

A comparative analysis of viral peptides presented by contemporary human and chimpanzee MHC class I molecules

Genetic factors such as the MHC influence the immunocompetence of an individual. MHC genes are the most polymorphic genes in primates, which is often interpreted as an adaptation to establish good T cell responses to a wide range of (evolving) pathogens. Chimpanzee MHC (Patr) genes are less polymorphic than human MHC (HLA) genes, which is surprising because chimpanzee is the older species of the two and is therefore expected to display more variation. To quantify the effect of the reduced polymorphism, we compared the peptide binding repertoire of human and chimpanzee MHC molecules. Using a peptide-MHC binding predictor and proteomes of >900 mammalian viruses, we show that, at the population level, the total peptide binding repertoire of Patr-A molecules is ~36% lower than that of their human counterparts, whereas the reduction of the peptide binding repertoire of the Patr-B locus is only 15%. In line with these results, different Patr-A molecules turn out to have largely overlapping peptide binding repertoires, whereas the Patr-B molecules are more distinct from each other. This difference is somewhat less apparent at the individual level, where we found that only 25% of the viruses are significantly better presented by "simulated" humans with heterozygous HLA-A and -B loci. Taken together, our results indicate that the Patr-B molecules recovered more after the selective sweep, whereas the Patr-A locus shows the most signs of the selective sweep with regard to its peptide binding repertoire.     

Phylogenetic analysis and structural predictions of human adenovirus penton proteins as a basis for tissue-specific adenovirus vector design

The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17'H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors.     

Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine

The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.     

A novel vector for a rapid generation of fiber-mutant adenovirus based on one step ligation and quick screening of positive clones

The generation of fiber-modified adenoviral vector has proven difficult. In the paper, we developed a new system for rapid construction of fiber-modified adenoviral vector containing foreign peptides in the HI loop or C-terminal of the fiber knob. The new system was established through the following processes. First, a unique BamHI mutation was made in the genome of Ad5 without causing amino acid change. Second, two unique restriction enzymes BamHI and SfuI, both with sticky end, were introduced in the HI loop or C-terminal of Ad5 fiber knob. Third, a lacza expression cassette was placed between BamHI and SfuI sites for a quick identification of positive cloning based on white-blue color screening. This system allows generation of recombinant adenoviral vector by a single step, in vitro ligation followed by quick white-color positive clone screening. To prove the principle of the method, Ad5HI-RGD by modifying HI-loop of the fiber knob with RGD motif and Ad5Cter-PK7 by modifying C-terminal of the knob with poly-lysine (pK7) were successfully generated in vitro. Ad5 with a knob modified in the HI loop of the fiber with Tat-PTD, NGR or SIKVAV peptide were also successfully developed. The transduction of the modified viruses for Hela, U87 MG and MDA-MB-231 cells was investigated in vitro compared with unmodified Ad5. In conclusion, the new vector system allows for a rapid generation of fiber-mutant adenovirus and provides useful tool for gene function analysis and cancer gene therapy.     

A comparative analysis of regulatory regions of the transthyretin gene in the mouse, human, and chimpanzee genomes

A full genome analysis of differences between the gene expression in the human and chimpanzee brains revealed that the gene for transthyretin, the carrier of thyroid hormones, is differently transcribed in the cerebella of these species. A 7-kbp DNA fragment of chimpanzee was sequenced to identify possible regulatory sequences responsible for the differences in expression. One hundred and thirteen substitutions were found in the chimpanzee sequence in comparison with the human sequence. About 40% of the substitutions were revealed within the repeating elements of the genome; their location and sizes did not differ from those in the corresponding fragments of the human genome, and the nucleotide sequences had a high degree of identity. A comparison of nucleotide sequences of the transthyretin region of human, chimpanzee, and mouse genes revealed substantial differences in the distribution of G + C content along the examined fragment in the human (chimpanzee) and mouse genes and allowed us to localize three sequence tracts with a higher degree of identity in the three species. One of these tracts is located in the promoter region of the gene, and the other two probably determine the specificity of transthyretin gene expression in the liver and brain. One of the conserved tracts of the chimpanzee genome was found to have a single and a triple nucleotide substitution. The triple substitution distinguishes chimpanzees from humans and mice, which have identical sequences of this site. It is likely that these substitutions are responsible for the differences in the expression levels of the transthyretin gene in the human and chimpanzee brains.     

A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines.

The tetracycline-dependent expression system has gained increasing popularity for the expression of any gene of interest. Careful choice of the expression vector has been suggested to exploit the full potential of this system. A novel tetracycline-sensitive expression vector based on a modified mouse mammary tumor virus promoter achieved considerably improved regulatory properties in a series of cell lines tested under transient and stable conditions. Therefore, the applicability of the tetracycline-dependent expression system can be largely enhanced by careful adaptation of the expression vector to the host cell line.     

A novel improved therapy strategy for diabetic nephropathy: Targeting AGEs

Diabetic nephropathy (DN), is a disorder that causes significant morbidity and mortality. Studies on the pathological mechanisms of DN reveal that advanced glycation end products (AGEs) play an important role in the pathogenesis of DN through interacting with receptors for advanced glycation end products (RAGE), which activate a series of intracellular signaling pathways. AGEs and RAGE have therefore been considered to be two potential key targets. Although multiple studies have been made for anti-DN therapy against AGEs or RAGE, the results have been disappointing due to poor effectiveness or to side effects in clinical practice. In this hypothesis article, we propose a novel treatment based on a dual-target approach. A kind of multi-functional intelligent nanoparticle is constructed, which has a core-shell nanoparticle structure to load the dual-target drugs (AGEs inhibitors and RAGE inhibitors), and has a functional "RAGE analog" to be used as "bait" to catch AGEs and target them to the kidney. Owing to its advantages of having a dual-target, synergistic effect and high efficiency, the proposition may have potential applications in DN therapy.     

A novel binary expression vector for production of human IL-10 in Escherichia coli and Bifidobacterium longum

A novel expression vector (pLR) driven by hup promoter and Bifidobacterium β-galactosidase signal peptide was constructed. The pLR vector was used for the expression of the optimized human IL-10 synthetic gene in Escherichia coli and Bifidobacterium longum. In both microorganisms, rhIL-10 was in a soluble form in total extract cells. The recombinant hIL-10 was partially processed in E. coli, whereas in Bifidobacterium all rhIL-10 was found in the mature form.     

Design of a novel type IV lipid-based delivery system for improved delivery of drugs with low partition coefficient

Abstract

Context: The physicochemical properties of drugs such as partition coefficient play a major role in the development of lipid-based drug delivery systems. The major obstacle lies in encapsulation of a drug with low partition coefficient into these systems.

Objective: The objective of this study was to design and optimize a novel lipid-based delivery system with higher loading, improved pharmacokinetics consequently enhancing the oral bioavailability of drugs with low partition coefficient like valsartan.

Materials and methods: The optimized formulation consists of Capryol 90, Cremophor RH 40, and Transcutol HP. Pseudo ternary phase diagrams were used to optimize the components and their concentrations in the formulation. Dissolution studies of the selected formulations were compared with plain drug and marketed product at three pH conditions (pH 1.2, 4.5 and 6.8). Pharmacokinetic parameters of optimized formulations were determined in Wistar rats and compared with that of plain drug.

Results and discussion: The optimized formulation with a mean particle size of 50?nm showed significant improvement (p?<?0.05) in dissolution rate with pH independence compared to plain drug and marketed product. The in vivo studies in Wistar rats revealed about 2.30- and 1.68-fold increase in the oral bioavailability and C_max of valsartan from lipid-based formulation compared to plain drug.

Conclusion: The engineered formulation strategy by type IV lipid-based formulations can be successfully exploited to improve the dissolution rate and oral deliverability of drugs like valsartan.     

A novel method for generating and screening peptides and libraries displayed on adenovirus fiber

Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre–lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The ‘acceptor’ vector does not contain the fiber gene, and therefore does not propagate until it has received a ‘donor’ fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.