The Experts below are selected from a list of 204 Experts worldwide ranked by ideXlab platform
Ernesto T A Marques - One of the best experts on this subject based on the ideXlab platform.
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Inverted Terminal Repeat sequences of adeno associated virus enhance the antibody and cd8 responses to a hiv 1 p55gag lamp dna vaccine chimera
Virology, 2004Co-Authors: Priya Chikhlikar, Luciana Barros De Arruda, Shikha Agrawal, Barry J Byrne, William B Guggino, Thomas J August, Ernesto T A MarquesAbstract:Abstract The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the Inverted Terminal Repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/ gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/ gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/ gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the “naked” DNA vaccines encoding the LAMP/ gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4 + T cell responses. In contrast, significantly higher levels of CD8 + and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG 1 isotype resulting from the activation of the Th2 subset of CD4+ T cells, that was sustained for at least 5 months after immunization.
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Inverted Terminal Repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera.
Virology, 2004Co-Authors: Priya Chikhlikar, Luciana Barros De Arruda, Shikha Agrawal, Barry J Byrne, William B Guggino, J. Thomas August, Ernesto T A MarquesAbstract:Abstract The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the Inverted Terminal Repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/ gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/ gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/ gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the “naked” DNA vaccines encoding the LAMP/ gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4 + T cell responses. In contrast, significantly higher levels of CD8 + and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG 1 isotype resulting from the activation of the Th2 subset of CD4+ T cells, that was sustained for at least 5 months after immunization.
Priya Chikhlikar - One of the best experts on this subject based on the ideXlab platform.
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Inverted Terminal Repeat sequences of adeno associated virus enhance the antibody and cd8 responses to a hiv 1 p55gag lamp dna vaccine chimera
Virology, 2004Co-Authors: Priya Chikhlikar, Luciana Barros De Arruda, Shikha Agrawal, Barry J Byrne, William B Guggino, Thomas J August, Ernesto T A MarquesAbstract:Abstract The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the Inverted Terminal Repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/ gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/ gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/ gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the “naked” DNA vaccines encoding the LAMP/ gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4 + T cell responses. In contrast, significantly higher levels of CD8 + and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG 1 isotype resulting from the activation of the Th2 subset of CD4+ T cells, that was sustained for at least 5 months after immunization.
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Inverted Terminal Repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera.
Virology, 2004Co-Authors: Priya Chikhlikar, Luciana Barros De Arruda, Shikha Agrawal, Barry J Byrne, William B Guggino, J. Thomas August, Ernesto T A MarquesAbstract:Abstract The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the Inverted Terminal Repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/ gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/ gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/ gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the “naked” DNA vaccines encoding the LAMP/ gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4 + T cell responses. In contrast, significantly higher levels of CD8 + and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG 1 isotype resulting from the activation of the Th2 subset of CD4+ T cells, that was sustained for at least 5 months after immunization.
Regine Heilbronn - One of the best experts on this subject based on the ideXlab platform.
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Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency.
The Journal of general virology, 2020Co-Authors: Daniela Hüser, Regine HeilbronnAbstract:Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus, AAVS1, on human chromosome 19 (chr19). To study the kinetics and frequency of chr19-specific integration, a rapid, sensitive and quantitative real-time PCR assay specific for AAV Inverted Terminal Repeat (ITR)-chr19 junction sequences was developed. Since the assay only detected right-hand AAV ITR-specific integration events, the development of a complementary left-hand ITR-specific real-time PCR assay is described. The time-course of left-hand ITR-dependent AAV integration at AAVS1 of chr19 was determined in AAV-2-infected HeLa cells. Both the kinetics and frequencies of left-hand ITR-dependent integration were found to be similar to those of the right-hand ITR. In addition, left-hand ITR-specific fusion sequences and chromosomal breakpoints within AAVS1 were variable, yet were the same as those found in right-hand ITR-chr19 junction sequences. Thus, the AAV-2 genome integrates site-specifically into chr19 with similar efficiency in either orientation.
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dna binding activity of adeno associated virus rep is required for Inverted Terminal Repeat dependent complex formation with herpes simplex virus icp8
Journal of Virology, 2012Co-Authors: Martin Alex, Stefan Weger, Mario Mietzsch, Heiko Slanina, Toni Cathomen, Regine HeilbronnAbstract:Herpes simplex virus (HSV) helper functions for (AAV) replication comprise HSV ICP8 and helicase-primase UL5/UL52/UL8. Here we show that N-Terminal amino acids of AAV Rep78 that contact the Rep-binding site within the AAV Inverted Terminal Repeat (ITR) are required for ternary-complex formation with infected-cell protein 8 (ICP8) on AAV single-strand DNA (ssDNA) in vitro and for colocalization in nuclear replication domains in vivo. Our data suggest that HSV-dependent AAV replication is initiated by Rep contacting the AAV ITR and by cooperative binding of ICP8 on AAV ssDNA.
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Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCR
Journal of Virology, 2002Co-Authors: Daniela Hüser, Stefan Weger, Regine HeilbronnAbstract:Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV Inverted Terminal Repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.
Geoffrey L Smith - One of the best experts on this subject based on the ideXlab platform.
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nucleotide sequence of 42 kbp of vaccinia virus strain wr from near the right Inverted Terminal Repeat
Journal of General Virology, 1991Co-Authors: Geoffrey L Smith, Sang Y Chan, Susan T HowardAbstract:The nucleotide sequence of 42090 bp of vaccinia virus strain WR is presented. The sequence includes the SalI L, F, G and I fragments and starts near the centre of the HindIII A fragment and extends rightwards towards the genomic terminus, finishing approximately 0·5 kb internal of the Inverted Terminal Repeat (ITR). Translation of this region has identified 65 open reading frames (ORFs) of greater than 65 amino acids in length. Fifty-one of these which do not extensively overlap other larger ORFs have been subjected to further analysis; the other 14 are termed minor ORFs. In the rightmost 28·7 kb, the genes are, with one exception, transcribed towards the genomic terminus, similar to the arrangement of genes at the left end of the virus genome. Internal of this region the genes are expressed off either DNA strand but still predominately rightwards. ORFs are tightly packed with few intergenic non-coding regions of greater than 250 bp. Protein sequence comparisons have established a remarkably high number of homologies with entries in existing protein databases. Of these, DNA ligase, thymidylate kinase, two serine-threonine protein kinases, two serine proteinase inhibitors (serpins), two interleukin-1 receptor homologues and a discontinuous ORF related to tumour necrosis factor receptor have been reported. Other homologies include lectins, profilin, 3β-hydroxy steroid dehydrogenase, superoxide dismutase, guanylate kinase, ankyrin and complement factor H. In addition, there are a number of polypeptides with predicted properties of membrane-associated, secretory or glyco-proteins. Twelve gene families are described here and elsewhere. There is considerable similarity between genes from the right and left end of the virus genome that may have arisen by Terminal transposition events. Several differences from the corresponding region of vaccinia virus strain Copenhagen sequence are noted. Near the right terminus the sequences diverge completely, and internal of this there are multiple examples of deletion of short sequences (eight to 10 nucleotides) that lie within penta- or hexanucleotide direct Repeats.
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vaccinia virus homologues of the shope fibroma virus Inverted Terminal Repeat proteins and a discontinuous orf related to the tumor necrosis factor receptor family
Virology, 1991Co-Authors: Geoffrey L Smith, Sang Y Chan, Susan T HowardAbstract:Abstract Nucleotide sequencing data from a region extending 35 kb inward from the right Inverted Terminal Repeat (ITR) of the vaccinia virus (VV) genome established the presence of VV homologues of the Shope fibroma virus (SFV) ITR proteins. The nucleotide sequences, comprising a total of 8.6 kb, and the amino acid translations for nine predicted open reading frames (ORFs) (designated SaIF4L, SaIF19R, SaIF21 R, B4R, B8R, B9R, BI OR, and 131 4R) are presented. Eight of the nine VV genes and all the SFV ORFs are transcribed towards their genomic termini. However, the relative positions of the VV genes (genus Orthopoxvirus) are different than those of the corresponding ORFs in SFV (genus Leporipoxvirus ), indicating complex rearrangements of DNA in the genome of one or both of these viruses subsequent to their divergence from a common ancestor. Several other features of the VV ORFs were noted. SaIF4L, B7R, B8R, and B9R have hydrophobic amino-Terminal signal sequences but lack discernible membrane anchor domains suggesting that the proteins may be secreted. VV ORF SaIF19R has a single cysteine-rich region homologous to the multiple domains of nerve growth factor receptor (NGFR), CD40, OX40 (a glycoprotein from the surface of activated murine T lymphocytes), and the recently described tumor necrosis factor receptors. Just downstream of the ORF SaIF19R and in a different reading frame, there are another two related cysteine-rich domains, indicating that SaIF19R was once a larger gene. B4R has homology to the host range gene of cowpox virus and to related genes near the opposite end of the vaccinia virus genome, and contains regions homologous to the Repeat domains of erythrocyte ankyrin. In addition, several of the VV ORFs have homology to ORFs from near the opposite end of the VV genome, thus increasing the number of known VV gene families.
Kenneth I. Berns - One of the best experts on this subject based on the ideXlab platform.
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the unusual properties of the aav Inverted Terminal Repeat
Human Gene Therapy, 2020Co-Authors: Kenneth I. BernsAbstract:Although the sequence of the AAV Inverted Terminal Repeat has been known for 40 years, there are still unanswered questions about functions attributable to the Terminal 125 nucleotides.
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The Unusual Properties of the AAV Inverted Terminal Repeat
Human Gene Therapy, 2020Co-Authors: Kenneth I. BernsAbstract:no abstract.
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a p5 integration efficiency element mediates rep dependent integration into aavs1 at chromosome 19
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Nicola J Philpott, Kenneth I. Berns, Janette Gomos, Erik FalckpedersenAbstract:Adeno-associated virus (AAV) undergoes site-specific integration into human chromosome 19 through a deletion-substitution mechanism at the well characterized AAVS1 site. We have shown previously that a cis element within the left end of the AAV genome enhances the efficiency of Rep-mediated site-specific integration into chromosome 19 when present in Inverted Terminal Repeat-containing recombinant AAV (rAAV) plasmids. We now demonstrate that a 138-bp cis element, the p5 integration efficiency element (p5IEE), mediates efficient integration. The p5IEE is not only required for efficient site-specific integration, it is also sufficient. Integration mediated by the p5IEE occurs in the absence of the AAV Inverted Terminal-Repeat elements. The data presented in this study demonstrate that the p5IEE is a multifunctional element, serving as the highly regulatable Rep promoter and the primary substrate for targeted integration.
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Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome.
Journal of Virology, 1995Co-Authors: Christophe Giraud, E Winocour, Kenneth I. BernsAbstract:A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-specific integration event in chromosome 19q13.3-qter (C. Giraud, E. Winocour, and K.I. Berns, Proc. Natl. Acad. Sci. USA 91:10039-10043, 1994). In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus). In most of the recombinants, one end of the viral genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration. Within this AAVS1 segment, the AAV insertion points were strikingly clustered around a binding site for the AAV regulatory protein. In all cases, the second junction with AAV occurred with vector DNA outside of the AAVS1 segment. With respect to the viral genome, one junction with the shuttle vector DNA occurred either within the AAV Inverted Terminal Repeat (itr), or near the P5 promoter, approximately 100 nucleotides distal to a modified itr. The modified itr in 5 of 11 recombinants involved a head-to-tail organization. In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a junction close to the P5 promoter; the AAV insert in this recombinant episome could be rescued by adenovirus infection and replicated to virus particles. The significance of the head-to-tail organization is discussed in terms of the possible circularization of AAV DNA before or during integration.
