Molecular Biotechnology (MOL BIOTECHNOL)

Publications in this journal

• Article: Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish.

  Noriko Umemoto, Yuhei Nishimura, Yasuhito Shimada, Yukiko Yamanaka, Seiya Kishi, Saki Ito, Kana Okamori, Yuuki Nakamura, Junya Kuroyanagi, Zi Zhang, Liqing Zang, Zhipeng Wang, Norihiro Nishimura, Toshio Tanaka
  [show abstract] [hide abstract]
  ABSTRACT: A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.
  Molecular Biotechnology 05/2013;
• Article: In Vivo Neutralization of Botulinum Neurotoxins Serotype E with Heavy-chain Camelid Antibodies (VHH).

  Hamid Bakherad, Seyed Latif Mousavi Gargari, Iraj Rasooli, Masoumeh Rajabibazl, Mohammad Mohammadi, Walead Ebrahimizadeh, Leila Safaee Ardakani, Hamed Zare
  [show abstract] [hide abstract]
  ABSTRACT: Ingestion of botulinum neurotoxin (BoNT) results in botulism, a severe and frequent fatal disease known in the world. Current treatments rely on antitoxins, such as equine antitoxin and human botulism immunoglobulin. In some cases, side effects have been reported, including early anaphylactic shock and late serum sickness. Thus, diagnosis and treatment measure of BoNT are necessary and crucial. In the present study, a single-domain variable heavy-chain (VHH) antibody fragment was obtained from an immune dromedary phage display library against the putative binding domain of botulinum neurotoxin E (BoNT/E), a non-toxic 50-kDa fragment. The characteristics of nanobody VHH include excellent production, superior heat stability and specific binding capacity to soluble antigen without cross-reaction to other relevant or irrelevant antigens. A total of 150 ng/Kg of nanobody entirely neutralized 3LD50 of the BoNT/E in an in vivo challenge of the mice. This phenomenon indicates BoNT/E toxin neutralizing capacity of the produced nanobody. These results also suggest possession of unique properties by the nanobody applicable in diagnostics or therapeutic purposes.
  Molecular Biotechnology 05/2013;
• Article: Glucose Metabolism, Hyperosmotic Stress, and Reprogramming of Somatic Cells.

  Rosalinda Madonna, Aniko Görbe, Peter Ferdinandy, Raffaele De Caterina
  [show abstract] [hide abstract]
  ABSTRACT: The availability of glucose and oxygen are important regulatory elements that help directing stem cell fate. In the undifferentiated state, stem cells, and their artificially reprogrammed equivalent-induced pluripotent stem cells (iPS) are characterized by limited oxidative capacity and active anaerobic glycolysis. Recent studies have shown that pluripotency-a characteristic of staminality-is associated with a poorly developed mitochondrial patrimony, while differentiation is accompanied by an activation of mitochondrial biogenesis. Besides being an important energy source in hypoxia, high glucose level results in hyperosmotic stress. The identification of specific metabolic pathways and biophysical factors that regulate stem cell fate, including high glucose in the extracellular medium, may therefore facilitate reprogramming efficiency and control the differentiation and fate of iPS cells, which are increasingly being explored as therapeutic tools. In this article, we review recent knowledge of the role of glucose metabolism and high glucose level as major anaerobic energy source, and a determinant of osmolarity as possible tools for reprogramming therapies in clinical applications. As in the diabetic setting hyperglycemia negatively affect the stem/progenitor cell fate and likely somatic reprogramming, we also discuss the in vivo potential transferability of the available in vitro findings.
  Molecular Biotechnology 05/2013;
• Article: Quantification of Rice Blast Disease Progressions Through Taqman Real-Time PCR.

  Mukhamad Su'udi, Jinyeong Kim, Jong-Mi Park, Shin-Chul Bae, Donghern Kim, Yong-Hwan Kim, Il-Pyung Ahn
  [show abstract] [hide abstract]
  ABSTRACT: Rice blast caused by Magnaporthe oryzae is a major disease in the paddy field and also a representative model system in the investigation of plant-microbe interactions. This study was undertaken to provide the quantitative evaluation method that specifically determines the amount of M. oryzae proliferation in planta. Real-time PCR was used as the detection strategy in combination with the primer pair and Taqman probe specific to MHP1, a unigene encoding HYDROPHOBIN that is indispensable for normal virulence expression. Based on the crossing point values from the PCR reactions containing a series of increasing concentration of cloned amplicon or fungal genomic DNA, correlation among the template's copy number or its amount and amplification pattern was calculated. Reliability of this equation was further confirmed using the DNA samples from the rice leaves infected with compatible or incompatible strains of M. oryzae. The primer pair used in the Taqman real-time PCR reaction can recognize the existence of fungal DNA as low as 1 pg. In sum, our quantitative evaluation system is applicable and reliable in the blast diagnosis and also in the estimation of objective blast disease progression.
  Molecular Biotechnology 05/2013;
• Article: Characterization of Zwitterionic Phosphatidylcholine-Based Bilayer Vesicles as Efficient Self-Assembled Virus-Like Gene Carriers.

  Reihaneh Ramezani, Majid Sadeghizadeh, Mehrdad Behmanesh, Saman Hosseinkhani
  [show abstract] [hide abstract]
  ABSTRACT: Entrapment of plasmid DNA (pDNA) in an aqueous compartment separated from the bulk external aqueous medium by a phospholipid bilayer resembles a structure similar to a primitive living cell, and interestingly, this phenomenon occurs completely self-assembled. Being inspired by such a structure as well as using the dehydration-rehydration technique, we were able to encapsulate pDNA without using multivalent cations and with high efficiency (98 %) into noncationic lipid bilayer vesicles. These liposomes which were composed of dimyristoyl-sn-glycero-3-phosphocholine unlike cationic liposomes, were nontoxic. The obtained liposome structure was able protect DNA against nuclease and was completely stable, in a way that even after 6 months, it still kept the pDNA in its structure, and there was a small change in its size (100-150 nm) determined by dynamic light scattering. The purpose of this research is to polarize the researchers' interest toward utilization of neutral liposomes originating from the cell membrane as the most efficient carrier for gene delivery. We indicated that in using such carriers, which are the most similar synthetic structures to viruses, their inability in electrostatic interaction with DNA would not be an obstacle for entrapping nucleic acids.
  Molecular Biotechnology 05/2013;
• Article: Heterologous Overexpression, Purification and Characterisation of an Alcohol Dehydrogenase (ADH2) from Halobacterium sp. NRC-1.

  Ann-Kathrin Liliensiek, Jennifer Cassidy, Gabriele Gucciardo, Cliadhna Whitely, Francesca Paradisi
  [show abstract] [hide abstract]
  ABSTRACT: Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.
  Molecular Biotechnology 05/2013;
• Article: Long Distance Multiple-Site Directed Plasmid Mutagenesis by One-Step PCR Using Non-overlapped Primers.

  Yuling Bu, Hui Wang, Jing Li, Zhaoxin Zhang, Yinghe Hu, Zhiliang Xu
  [show abstract] [hide abstract]
  ABSTRACT: Site-directed mutagenesis is a very important technique in molecular biological researches. We have developed a new method for long distance multiple-site plasmid mutation by one-step PCR using non-overlap primers. These primers were carefully designed and contained desired mutations in the middle of the primers flanked with 18-25 bp of correct sequence. One pair of the primers was able to generate a short megaprimer. Decreases in the concentrations of these primers increased efficiency of the multiple-site plasmid mutation. All of the mutant PCRs were performed at a common annealing temperature at 55 °C. This method could be widely used in all multiple-site plasmid mutations.
  Molecular Biotechnology 05/2013;
• Article: Construction of an Expression Vector for Production and Purification of Human Somatostatin in Escherichia coli.

  Sergi Maicas, Ismaïl Moukadiri, Almudena Nieto, Eulogio Valentín
  [show abstract] [hide abstract]
  ABSTRACT: Somatostatin/growth hormone-inhibiting hormone is the peptide that inhibits secretion of somatotropin/growth hormone. Solid-phase synthesis methods are being currently used to produce somatostatin. Recombinant peptide synthesis is widely described for the production of small proteins and peptides; however, the production at industrial scale of peptides for biopharmaceutical applications is limited for economic reasons. Here, we propose the use of a new pGB-SMT plasmid to produce Somatostatin, as a C-terminal fusion protein with a Kluyveromyces lactis β-galactosidase fragment. To facilitate removal of that fragment by CNBr cleavage, a methionine residue was introduced at the N-terminal of the hormone peptide. The use of this construction enables an IPTG-free expression system. The suitability of this procedure has been assessed in a 15 l scale-up experiment yielding almost 300 mg, with purity >99 % and it is being implemented for commercial scale. The plasmid pGB-SMT here described is an alternative option for a cheap and high expression of other short peptide hormones.
  Molecular Biotechnology 05/2013;
• Article: Reprogrammed Peripheral Blood Mononuclear Cells are Able to Survive Longer in Irradiated Female Mice.

  Guang-Ping Ruan, Yi-Bing Han, Guang-Hong Ruan, Xiang-Qing Zhu, Xiang Yao, Rong-Qing Pang, Xue-Ming Cai, Jin-Xiang Wang, Jie He, Jing Zhao, Guang-Xu Zhu, Xin-Ming Xu, Xing-Hua Pan
  [show abstract] [hide abstract]
  ABSTRACT: Induced multipotent stem (iMS) cells are originated from somatic cells and become multipotent by genetic and/or epigenetic modifications. Previous studies have shown that the fish oocytes extracts (FOE) can induce skin fibroblast cells into iMS cells. In this study, we aim to determine whether FOE can similarly induce mouse peripheral blood mononuclear cells (PBMCs) into the iMS state and if so, whether they can survive longer when they are transplanted into the irradiation female mice. PBMCs of GFP-transgenic male mice were cultured and transiently reprogrammed by FOE. They were deemed reaching the iMS state after detection of expression of stem cell markers. The iMS-like PBMCs were transplanted into female C57BL mice by tail vein injection. The spleen wet weights as well as numbers of colonies of the recipient mice were examined. The results showed the spleen wet weights and numbers of spleen colonies of FOE-induced group were all significantly higher than those of the non-induced group and negative control group. On day 90 after transplantation, FISH analysis detected the presence of Y chromosome in the induced group, but not of the other groups. The current findings demonstrate that FOE-induced PBMCs are able to survive longer in irradiated female mice.
  Molecular Biotechnology 05/2013;
• Article: Alternative Promoters Regulate Cold Inducible RNA-Binding (CIRP) Gene Expression and Enhance Transgene Expression in Mammalian Cells.

  Mohamed B Al-Fageeh, C Mark Smales
  [show abstract] [hide abstract]
  ABSTRACT: The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well characterized; however, the equivalent cold stress responses are not. In particular, the transcriptional mechanisms that lead to enhanced gene-specific expression upon cold stress have yet to be elucidated. We report here in silico and experimental identification and characterization of transcriptional control elements that regulate cold inducible RNA-binding (CIRP) gene expression and demonstrate these can be used for enhanced transgene expression. In silico analysis identified the core CIRP promoter and a number of conserved transcription factor-binding sites across mammalian species. The core promoter was confirmed by experimental studies that located the basal transcriptional regulatory elements of CIRP within 264 nucleotides upstream of the transcription start site. Deletion analysis of a fragment from -264 to -64 that contained two putative CAAT-binding sites abolished promoter activity. A second promoter was identified in the region -452 to -264 of the transcription start site which was able to drive transcription independent of the core promoter. As the two CIRP promoters were transcriptionally active and possibly cold responsive, we used electrophoretic mobility shift assays to show that both promoter regions are able to bind factors within a nuclear extract in a dose-dependent manner and that the formation of these complexes was specific to the promoter regions. Finally, we successfully demonstrate using a reporter gene approach that enhanced transgene expression can be achieved using the identified CIRP promoter.
  Molecular Biotechnology 04/2013;
• Article: Molecular Strain Typing of Brucella abortus Isolates from Italy by Two VNTR Allele Sizing Technologies.

  Riccardo De Santis, Massimo Ancora, Fabrizio De Massis, Andrea Ciammaruconi, Katiuscia Zilli, Elisabetta Di Giannatale, Valentina Pittiglio, Silvia Fillo, Florigio Lista
  [show abstract] [hide abstract]
  ABSTRACT: Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.
  Molecular Biotechnology 04/2013;
• Article: Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences.

  Juliane Strien, Juliane Sanft, Gita Mall
  [show abstract] [hide abstract]
  ABSTRACT: PCR is a commonly used and highly efficient technique in biomolecular laboratories for specific amplification of DNA. However, successful DNA amplification can be very time consuming and troublesome because many factors influence PCR efficiency. Especially GC-rich DNA complicates amplification because of generation of secondary structures that hinder denaturation and primer annealing. We investigated the impact of previously recommended additives such as dimethylsulfoxide (DMSO), magnesium chloride (MgCl2), bovine serum albumin (BSA), or formamide. Furthermore, we tested company-specific substances as Q-Solution, High GC Enhancer, and Hi-Spec; various actively promoted polymerases as well as different PCR conditions for their positive effects on DNA amplification of templates with moderate and extremely high CG-content. We found considerable differences of specificity and quantity of product between different terms. In this article, we introduce conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates.
  Molecular Biotechnology 04/2013;
• Article: Systematic Optimization of Multiplex Zymography Protocol to Detect Active Cathepsins K, L, S, and V in Healthy and Diseased Tissue: Compromise Among Limits of Detection, Reduced Time, and Resources.

  Jerald E Dumas, Manu O Platt
  [show abstract] [hide abstract]
  ABSTRACT: Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.
  Molecular Biotechnology 03/2013;
• Article: A Comparative Structural and Bioanalytical Study of IVIG Clinical Lots.

  Annamaria Sandomenico, Valeria Severino, Angela Chambery, Annalia Focà, Giuseppina Focà, Claudio Farina, Menotti Ruvo
  [show abstract] [hide abstract]
  ABSTRACT: Intravenous immunoglobulin are important bio-therapeutics used in the replacement therapy for primary and secondary immunodeficiencies, chronic inflammatory disorders and several autoimmune haematologic disorders. Currently, a number of immunoglobulin intravenous (IVIG) products have been approved by the Food and Drug Administration (FDA) and are available commercially. It is known that small differences in the manufacturing processes as well as in the formulations may affect their clinical efficacy and tolerability. Therefore, given the complexity of the multi-step process required for the isolation of IVIG from human plasma, it is necessary to ensure a rigorous quality control of final products. We show here that a set of different bioanalytical techniques can be conveniently used to comparatively characterize, at a quantitative and qualitative level, different lots of IVIG preparations and to unveil randomly occurring impurities which can also affect the overall product stability. We have used circular dichroism, surface plasmon resonance and two-dimensional electrophoresis (2DE), and have demonstrated that this combination of bioanalytical approaches is very useful to improve the quality control of antibodies and to monitor the reliability of the IVIG manufacturing process.
  Molecular Biotechnology 03/2013;
• Article: Validation Assay of p3_VvAGL11 Marker in a Wide Range of Genetic Background for Early Selection of Stenospermocarpy in Vitis vinifera L.

  Carlo Bergamini, Maria Francesca Cardone, Angelo Anaclerio, Rocco Perniola, Arianna Pichierri, Rosalinda Genghi, Vittorio Alba, Lucia Rosaria Forleo, Angelo Raffaele Caputo, Cinzia Montemurro, Antonio Blanco, Donato Antonacci
  [show abstract] [hide abstract]
  ABSTRACT: DNA markers technology, derived from research in molecular biology and genomics, offers great promise for plant breeding, allowing the "molecular breeding" via marker-assisted selection. Grapevine genomic resources allowed, in recent years, the characterization at molecular level of genes involved in interesting phenotypes such as stenospermocarpic seedlessness, a trait really appreciated by consumers. Recent studies in table grapes revealed that the VvAGL11 gene, member of the D-lineage MADS-box family, controls the ovule identity, and thus potentially playing an important role in stenospermocarpy. Intragenic markers of VvAGL11 have been found and tested for breeding purposes. In the present paper, we describe an in deep assay on a total of 475 genotypes derived by our own grape germplasm and seeded × seedless crosses F1 offspring, to evaluate and verify the "diagnostic" power of VvAGL11 in marker-assisted selection. We found only 8/475 that were seeded and carried the seedless-associated allele in the STS p3_VvAGL11. However, and most importantly, there were no seedless varieties without such allele. We validated the marker as a 100 % effective tool for early negative selection of stenospermocarpy in Vitis vinifera L. crosses.
  Molecular Biotechnology 03/2013;
• Article: A Comparative Study of Nitrilases Identified by Genome Mining.

  Ondřej Kaplan, Alicja B Veselá, Alena Petříčková, Fabrizia Pasquarelli, Martina Pičmanová, Anna Rinágelová, Tek Chand Bhalla, Miroslav Pátek, Ludmila Martínková
  [show abstract] [hide abstract]
  ABSTRACT: Escherichia coli strains expressing different nitrilases transformed nitriles or KCN. Six nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetonitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising nitrilases for the transformation of industrially important substrates were found: the nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the nitrilases from N. crassa and A. niger for (R,S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine.
  Molecular Biotechnology 03/2013;
• Article: Data Mining in Proteomics: From Standards to Applications – Methods in Molecular Biology Vol. 696 (Book Review).

  Luisa Rusconi
  Molecular Biotechnology 03/2013; 53:351.
• Article: Development and Padronization of Three Multiplex PCRs for the Diagnosis of Chlamydia trachomatis, Toxoplasma gondii, Herpes Simplex Viruses 1 and 2, and Cytomegalovirus.

  Danielle A G Zauli, Carla Lisandre Paula de Menezes, Cristiane Lommez de Oliveira
  [show abstract] [hide abstract]
  ABSTRACT: To develop multiplex PCRs (mPCRs) that allows simultaneous diagnosis of the infectious agents Chlamydia trachomatis, Toxoplasma gondii, HSV 1/2, and Cytomegalovirus (CMV). The study included patients with clinical suspicion of these agents, and clinical samples were blood, cerebrospinal fluid, urine, vaginal swabs, and amniotic fluid. After the extraction of DNA, this was used as a template in amplification by PCR of selected genes. The following conditions were tested: primer concentration, MgCl(2) concentration, and annealing temperature. Three mPCRs were developed: multiplex I (CMV, HSV 1/2), multiplex II (CMV, HSV 1/2, T. gondii), and multiplex III (C. trachomatis, T. gondii, HSV 1/2, and CMV). The primer pairs used were shown to be specific for each infectious agent, and the specificity of mPCR assays was 100 %. Both the reactions of the monoplex PCR and mPCR produced a detection limit of 2 × 10(-5) to 6 × 10(-7) ng/μl of different DNAs. Upon conclusion, amplified products of expected size were obtained in 3 different reactions, and all the infectious agents were detected simultaneously in each mPCR. The concordant results of the study suggest that mPCR can be a powerful tool to improve the diagnostics of infectious diseases.
  Molecular Biotechnology 02/2013;
• Article: Expression of Human Rotavirus Chimeric Fusion Proteins from Replicating but Non Disseminating Adenovectors and Elicitation of Rotavirus-Specific Immune Responses in Mice.

  Aurélie Girard, Elodie Roques, Marie-Claude St-Louis, Bernard Massie, Denis Archambault
  [show abstract] [hide abstract]
  ABSTRACT: The aim of this study was to evaluate the usefulness of replicating but non disseminating adenovirus vectors (AdVs) as vaccine vector using human rotavirus (HRV) as a model pathogen. HRV VP7, VP4, or VP4Δ (N-terminal 336 amino acids of VP4) structural proteins as well as the VP4Δ::VP7 chimeric fusion protein were expressed in mammalian cells when delivered with the AdVs. A preliminary experiment demonstrated that VP4Δ was able to induce a HRV-specific IgG response in BALB/c mice inoculated intramuscularly with AdVs expressing the rotaviral protein. Moreover, an AdV-prime/plasmid DNA-boost regimen of vectors resulted in VP4Δ-specific antibody (Ab) titers ~4 times higher than those obtained from mice immunized with AdVs alone. Subsequently, the various HRV protein-encoding AdVs were compared using the AdV-prime/plasmid DNA-boost regimen. Higher IgG and IgA responses to HRV were obtained when VP4Δ::VP7 fusion protein was used as an immunogen as compared to VP7 or VP4 alone or to a mix of both proteins delivered independently by AdVs. A synergetic effect in terms of Ab was obtained with VP4Δ::VP7. In conclusion, this study demonstrated for the first time the suitability of using replicating but non disseminating AdVs as vaccine vector and the VP4Δ::VP7 fusion protein as an immunogen for vaccination against HRV.
  Molecular Biotechnology 02/2013;