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At least 307 records · Page 17Linked to original sources
PubMed2002

[Expression of the main structural antigen VP6 of human rotavirus by recombinant adenovirus and immune responses induced in vivo].

BACKGROUND: Constructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo. METHODS: The cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA. Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot. BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus. RESULTS: Recombinant adenovirus named rvAd-VP6 was obtained. The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably. The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively. In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100. Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group. The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation. CONCLUSIONS: The recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.

Adenoviridae↗
PubMed2009

Directed evolution of adeno-associated virus for glioma cell transduction.

Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.

Capsid Proteins↗
PubMed1998

New generation of African horse sickness virus vaccines based on structural and molecular studies of the virus particles.

African horse sickness virus (AHSV) is a member of the genus Orbivirus, which also includes bluetongue virus (BTV) and epizootic haemorrhagic disease (EHDV) virus. These orbiviruses have similar morphological and biochemical properties, with distinctive pathobiological properties and host ranges. Sequencing studies of the capsid proteins have revealed evolutionary relationships between these viruses. Biochemical studies of the viruses together with the expression of individual proteins and protein complexes have resulted in the development of new generation vaccines. Baculovirus expressed AHSV VP2 provides protection against death caused by AHSV challenge. Similarly, BTV VP2 alone elicits protective neutralising antibodies against BTV in sheep, which is enhanced in the presence of VP5. Recent developments in biotechnology (multiple gene expression baculovirus systems) have made it possible to synthesise orbivirus particles that biochemically and immunologically mimic authentic virions but lack the genetic material. Particle doses as low as 10 micrograms elicit responses that are sufficient to protect sheep 15 months post vaccination, against virulent virus challenge. Moreover, knowledge of the three dimensional structure of these particles enables us to engineer them to deliver multiple foreign peptide components representing other viral epitopes (e.g. foot and mouth disease virus and influenza virus) in order to elicit protective immunity.

African Horse Sickness Virus↗
PubMed1997

Established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity.

Preclinical arterial gene transfer studies with adenoviral vectors are typically performed in laboratory animals that lack immunity to adenovirus. However, human patients are likely to have prior exposures to adenovirus that might affect: (a) the success of arterial gene transfer; (b) the duration of recombinant gene expression; and (c) the likelihood of a destructive immune response to transduced cells. We confirmed a high prevalence (57%) in adult humans of neutralizing antibodies to adenovirus type 5. We then used a rat model to establish a central role for the immune system in determining the success as well as the duration of recombinant gene expression after adenovirus-mediated gene transfer into isolated arterial segments. Vector-mediated recombinant gene expression, which was successful in naive rats and prolonged by immunosuppression, was unsuccessful in the presence of established immunity to adenovirus. 4 d of immunosuppressive therapy permitted arterial gene transfer and expression in immune rats, but at decreased levels. Ultraviolet-irradiated adenoviral vectors, which mimic advanced-generation vectors (reduced viral gene expression and relatively preserved capsid function), were less immunogenic than were nonirradiated vectors. A primary exposure to ultraviolet-irradiated (but not nonirradiated) vectors permitted expression of a recombinant gene after redelivery of the same vector. In conclusion, arterial gene transfer with current type 5 adenoviral vectors is unlikely to result in significant levels of gene expression in the majority of humans. Both immunosuppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers to adenovirus-mediated arterial gene transfer.

Adenoviridae Infections↗
PubMed2003

Generation of fiber-modified adenovirus vectors containing heterologous peptides in both the HI loop and C terminus of the fiber knob.

BACKGROUND: Fiber-modified adenovirus (Ad) vectors can be effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Several types of fiber-modified Ad vectors have been developed. In this study, we evaluated the functionality of several fiber-modified Ad vectors. METHODS: We developed a simple method based on in vitro ligation to construct Ad vectors containing heterologous foreign peptides in both the HI loop and C terminus of the fiber knob. A functional comparison of Ad vectors containing RGD and/or K7 (KKKKKKK) peptide in the HI loop or C terminus of the fiber knob was performed in several types of human, mouse, and rat cells, including CAR-positive and -negative cells, and tumor cells in mice in vivo. RESULTS: In the case of the in vitro experiment, Ad vectors containing RGD peptide in the HI loop of the fiber knob showed a higher level of gene transfer than vectors containing RGD peptide at the C terminus of the fiber knob. Ad vectors containing K7 peptide at the C terminus of the fiber knob showed levels of gene transfer similar to those of Ad vectors containing RGD peptide in the HI loop of the fiber knob, depending on the cell type. Ad vectors containing both peptides in the HI loop or C terminus of the fiber knob showed the highest levels of gene transfer and a broader tropism. For gene transfer into tumor cells in vivo, the Ad vectors containing RGD peptide were the most efficient. CONCLUSIONS: In the experiment using cultured cells, Ad vectors containing both RGD and K7 peptides were the most efficient with a broader tropism. In contrast, in the experiment in vivo, Ad vectors containing RGD peptide in the HI loop of the fiber knob were more efficient than the vectors containing K7 peptide (including double-modified vectors containing both the RGD and K7 peptides). These comparative analyses could provide a systemic reference for the use of fiber-modified Ad vectors. Our simple method, in which the peptide of interest can be expressed in Ad vectors in either the HI loop or the C terminus of the fiber knob, or both, could be a powerful tool for gene transfer into mammalian cells in studies of gene function as well as in gene therapy.

Adenoviridae↗
PubMed2003

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle.

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.

Amino Acid Sequence↗
PubMed2015

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

Dependovirus↗
PubMed2003

Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus.

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter. Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells. Approximately 4500 yeast deletion strains ( approximately 80% of yeast genes) were screened in duplicate and selected strains analyzed further. This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold. Several of these genes were shown previously to function in BMV replication, validating the approach. Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function. The results further illuminate virus and cell pathways. Further refinement of virus screening likely will reveal contributions from additional host genes.

Bromovirus↗
PubMed1988

A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing.

Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor. The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein. All known or predicted cleavage sites in the TEV polyprotein are flanked by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Gly dipeptide. By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site. The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase. Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis. Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.

Amino Acid Sequence↗
PubMed2004

Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity.

The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.

Animals↗
PubMed1993

Production of human antibodies using bacteriophage.

The immune system produces antibodies by a process of antigen-driven selection. An in vitro process of antigen-driven selection, based on the display of antibody fragments on filamentous bacteriophage, has recently been developed. This enables human antibody fragments of high affinity and specificity to be produced without immunization.

Animals↗
PubMed2006

Virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes.

The selection and assembly of materials are central issues in the development of smaller, more flexible batteries. Cobalt oxide has shown excellent electrochemical cycling properties and is thus under consideration as an electrode for advanced lithium batteries. We used viruses to synthesize and assemble nanowires of cobalt oxide at room temperature. By incorporating gold-binding peptides into the filament coat, we formed hybrid gold-cobalt oxide wires that improved battery capacity. Combining virus-templated synthesis at the peptide level and methods for controlling two-dimensional assembly of viruses on polyelectrolyte multilayers provides a systematic platform for integrating these nanomaterials to form thin, flexible lithium ion batteries.

Bacteriophage M13↗
PubMed1996

Strategy for producing new foot-and-mouth disease vaccines that display complex epitopes.

Widely used inactivated vaccines for foot-and-mouth disease (FMD) induce protective immunity, but vaccine production plants and residual virus in the vaccine itself have been implicated in disease outbreaks. The structure of the FMD virion has been determined, and although much of the surface of the viral particle is produced by complex folding of the three surface-exposed capsid proteins (VP1-3), some surface regions representing important linear epitopes can be mimicked by recombinant proteins or synthetic peptides. Vaccine candidates based on these products stimulate immune responses to foot-and-mouth virus (FMDV), but do not always protect livestock from disease. The basis of protective immunity to FMDV has been explored using genetic engineering to produce antigenic chimeras of the virus. Studies with these chimeras have shown that a strong and protective immune response can be generated in livestock to epitopes outside the sequential epitopes incorporated into previous subunit vaccine candidates. Genetic engineering of the virus has also been used to demonstrate that changes within the sequence encoding an arginine-glycine-aspartic acid (RGD) sequence in VP1 abrogate virus binding to cells in culture, confirming the role of RGD as the receptor binding site. Based on this information, genetically stable viruses which cannot bind to cells have been created by deleting the nucleotides coding the RGD sequence. The receptor binding site-deleted viruses have been shown to be non-infectious in tissue culture, mice, and swine. Cattle vaccinated with these viruses are protected from disease when challenged with virulent FMDV, demonstrating that they could serve as the basis for safer FMD vaccines.

Amino Acid Sequence↗
PubMed1992

Modulation of T-cell reactivity to synthetic peptide analogues of foot-and-mouth disease virus in sheep by amino acid substitutions.

The ability of synthetic peptide analogues of foot-and-mouth disease virus VP1 capsid protein to induce T-cell proliferation in vitro following immunization of sheep with the uncoupled peptides was assessed. Elevated T-cell responses were obtained to a 21-residue peptide containing VP1 residues 141-158, and a 40-residue peptide containing residues 200-213 and 141-158 linked via a diproline-serine spacer. In contrast, no significant T-cell response was obtained with a 19-residue peptide containing residues 200-213 alone. In an attempt to engineer T-cell reactivity to this peptide, a sequence motif found in many peptides recognized by human or mouse T-cells was introduced by amino acid substitution. Substitution of a glycine or an aspartic acid for an alanine at position 207 in the 19-residue peptide resulted in the introduction of two such motifs running consecutively. Immunization of sheep with these peptides resulted in significant T-cell proliferative responses and elevated antibody responses. Analysis of further sequence variants showed that T-cell responsiveness was maintained with peptides containing single amino acid changes within these motifs, provided position 207 was glycine. The results thus suggest that increased T-cell reactivity, might be engineered via sequence manipulation of the 200-213 component of the 40-residue synthetic peptide. Such an additional T-cell epitope in the 40-residue peptide could potentially result in superior neutralizing antibody responses directed against the major epitope in residues 141-160 of VP1.

Amino Acid Sequence↗
PubMed1999

A disulfide-bound HIV-1 V3 loop sequence on the surface of human rhinovirus 14 induces neutralizing responses against HIV-1.

An immunogenic sequence from the V3 loop of the MN isolate of human immunodeficiency virus type 1 (HIV-1), His-Ile-Gly-Pro-Gly-Arg-Ala-Phe, was transplanted onto a surface loop of the VP2 capsid protein of human rhinovirus 14. To optimize for virus viability and immunogenicity of the transplanted sequence, the HIV sequence was flanked by (1) a cysteine residue that could form a disulfide bond and (2) randomized amino acids (in either of two arrangements) to generate numerous presentations of the Cys-Cys loop. The location for engineering in VP2 was chosen by searching the geometries of disulfide-bound loops in known protein structures. A model for the structure of the transplanted V3 loop sequence was developed using molecular dynamics and energy minimization calculations. Proteolytic digestion with and without reducing agent demonstrated the presence of the disulfide bond in the chimeric virus examined. Monoclonal and polyclonal antibodies directed against the V3 region of the HIV-1MN strain potently neutralized two chimeric viruses. Guinea pig antisera against two chimeric viruses were able to neutralize HIV-1MN and HIV-1ALA-1 in cell culture. The ability of chimeric viruses to elicit antibodies capable of neutralizing the source of the transplanted sequence could be favorable for vaccine development.

Animals↗
PubMed2006

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain.

Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.

Animals↗
PubMed2005

Strategies for the prevention of cervical cancer by human papillomavirus vaccination.

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed.

Antibodies, Viral↗
PubMed2002

A minimized M13 coat protein defines the requirements for assembly into the bacteriophage particle.

The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core. P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process. A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat. In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix. The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes. In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm. These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus. We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes. The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat. The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.

Bacteriophage M13↗