scholarly journals Adeno-Associated Virus Antibody Profiles in Newborns, Children, and Adolescents

2011 ◽  
Vol 18 (9) ◽  
pp. 1586-1588 ◽  
Author(s):  
Roberto Calcedo ◽  
Hiroki Morizono ◽  
Lili Wang ◽  
Robert McCarter ◽  
Jianping He ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Children And Adolescents ◽  
Neutralizing Antibodies ◽  
Human Subjects ◽  
Natural Infection ◽  
Childhood And Adolescence ◽  
Wild Type ◽  
Virus Antibody ◽  
Aav Vector ◽  
Adeno Associated Virus

ABSTRACTNeutralizing antibodies (NAb) to an adeno-associated virus (AAV) vector due to previous natural infection with wild-type AAV can significantly limit gene transfer. NAb titers to AAV serotype 2 (AAV2) and AAV8 in human subjects (0 to 18 years) were studied. NAb prevalence is moderate at birth, decreases markedly from 7 to 11 months, and then progressively increases through childhood and adolescence.

Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3532-3532
Author(s):  
Stephan Maersch ◽  
Anke Huber ◽  
Michael Hallek ◽  
Hildegard Buening ◽  
Luca Perabo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Transfer ◽  
Directed Evolution ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Multiple Point ◽  
Repeated Treatment ◽  
Therapeutic Gene ◽  
Adeno Associated Virus

Abstract Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.

scholarly journals Site-Specific Integration of an Adeno-Associated Virus Vector Plasmid Mediated by Regulated Expression of Rep Based on Cre-loxP Recombination

2000 ◽  
Vol 74 (22) ◽  
pp. 10631-10638 ◽  
Author(s):  
Wataru Satoh ◽  
Yukihiko Hirai ◽  
Kenji Tamayose ◽  
Takashi Shimada
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Wild Type ◽  
Chromosome 19 ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Coding Sequence ◽  
Site Specific Integration ◽  
Regulated Expression

ABSTRACT Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5′ end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

Dose response results of self complementary adeno-associated virus (AAV) vector-mediated factor IX gene transfer in non-human primates

Haemophilia ◽  
2009 ◽  
Vol 15 (2) ◽  
pp. 635-635
Author(s):  
ULRIKE REISS ◽  
ANDREW DAVIDOFF ◽  
JOHN GRAY ◽  
ARTHUR NIENHUIS ◽  
AMIT NATHWANI
Keyword(s):  
Gene Transfer ◽  
Dose Response ◽  
Factor Ix ◽  
Aav Vector ◽  
Adeno Associated Virus

scholarly journals Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

2002 ◽  
Vol 76 (4) ◽  
pp. 1904-1913 ◽  
Author(s):  
Chunping Qiao ◽  
Juan Li ◽  
Anna Skold ◽  
Xudong Zhang ◽  
Xiao Xiao
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cost Effective ◽  
Helper Virus ◽  
Vector System ◽  
Wild Type ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Packaging Cell ◽  
Producer Cell

ABSTRACT The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

scholarly journals Circulating Anti-Wild-Type Adeno-Associated Virus Type 2 (AAV2) Antibodies Inhibit Recombinant AAV2 (rAAV2)-Mediated, but Not rAAV5-Mediated, Gene Transfer in the Brain

2004 ◽  
Vol 78 (12) ◽  
pp. 6344-6359 ◽  
Author(s):  
Carmen S. Peden ◽  
Corinna Burger ◽  
Nicholas Muzyczka ◽  
Ronald J. Mandel
Keyword(s):  
Immune Response ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Natural Infection ◽  
Cell Counting ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Live Virus ◽  
The Brain

ABSTRACT Epidemiological studies report that 80% of the population maintains antibodies (Ab) to wild-type (wt) adeno-associated virus type 2 (AAV2), with 30% expressing neutralizing Ab (NAb). The blood-brain barrier (BBB) provides limited immune privilege to brain parenchyma, and the immune response to recombinant AAV (rAAV) administration in the brain of a naive animal is minimal. However, central nervous system transduction in preimmunized animals remains unstudied. Vector administration may disrupt the BBB sufficiently to promote an immune response in a previously immunized animal. We tested the hypothesis that intracerebral rAAV administration and readministration would not be affected by the presence of circulating Ab to wt AAV2. Rats peripherally immunized with live wt AAV2 and naive controls were tested with single intrastriatal injections of rAAV2 encoding human glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Striatal readministration of rAAV2-GDNF was also tested in preimmunized and naive rats. Finally, serotype specificity of the immunization against wt AAV2 was examined by single injections of rAAV5-GFP. Preimmunization resulted in high levels of circulating NAb and prevented transduction by rAAV2 as assessed by striatal GDNF levels. rAAV2-GFP striatal transduction was also prevented by immunization, while rAAV5-GFP-mediated transduction, as assessed by stereological cell counting, was unaffected. Additionally, inflammatory markers were present in those animals that received repeated administrations of rAAV2, including markers of a cell-mediated immune response and cytotoxic damage. A live virus immunization protocol generated the circulating anti-wt-AAV Ab seen in this experiment, while human titers are commonly acquired via natural infection. Regardless, the data show that the presence of high levels of NAb against wt AAV can reduce rAAV-mediated transduction in the brain and should be accounted for in future experiments utilizing this vector.

scholarly journals Successful Readministration of Adeno-Associated Virus Vectors to the Mouse Lung Requires Transient Immunosuppression during the Initial Exposure

1998 ◽  
Vol 72 (12) ◽  
pp. 9795-9805 ◽  
Author(s):  
Christine L. Halbert ◽  
Thomas A. Standaert ◽  
Christopher B. Wilson ◽  
A. Dusty Miller
Keyword(s):  
Neutralizing Antibodies ◽  
Marker Gene ◽  
Airway Epithelial Cells ◽  
Mouse Lung ◽  
Similar Response ◽  
Alveolar Cells ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Initial Exposure ◽  
Immunodeficient Mice

ABSTRACT The airway is an important target for gene transfer to treat cystic fibrosis and other diseases that affect the lung. We previously found that marker gene expression did not persist in the bronchial epithelium following adeno-associated virus (AAV) vector administration to the rabbit lung. In an attempt to promote continued expression, we tested repeat vector administration, but no additional transduction was observed, and the block to transduction correlated with the appearance of neutralizing antibodies to the viral capsid. Here we show that mice exhibit a similar response but that treatment with anti-CD40 ligand antibody (MR1) and a soluble CTLA4-immunoglobulin fusion protein (CTLA4Ig) at the time of primary AAV vector exposure allowed successful repeat transduction and prevented production of neutralizing antibodies. We also tested the possibility that an immune response caused the loss of marker-positive cells in the epithelial population in rabbits by evaluating AAV vector expression in immunocompetent and immunodeficient mice. In contrast to results in rabbits, marker protein expression persisted in the lung in both groups of mice. AAV vector transduction occurred in alveolar cells, airway epithelial cells, and smooth muscle cells, and vector expression persisted for at least 8 months. Although data on persistence of AAV vector expression in the human lung are not available, it is likely that repeat transduction will be necessary either due to loss of expression or to the need for repeat administration to deliver effective amounts of AAV vectors. Results presented here indicate that transient immunosuppression will allow such repeat vector treatment of the lung.

scholarly journals Hybrid Vectors Based on Adeno-Associated Virus Serotypes 2 and 5 for Muscle-Directed Gene Transfer

2001 ◽  
Vol 75 (13) ◽  
pp. 6199-6203 ◽  
Author(s):  
Markus Hildinger ◽  
Alberto Auricchio ◽  
Guangping Gao ◽  
Lili Wang ◽  
Narendra Chirmule ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Human Subjects ◽  
Large Population ◽  
Previous Exposure ◽  
Healthy Human ◽  
Adeno Associated Virus ◽  
Hybrid Vectors ◽  
Healthy Human Subjects ◽  
Cross Neutralization

ABSTRACT Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.

scholarly journals HVEM signaling promotes protective antibody-dependent cellular cytotoxicity (ADCC) vaccine responses to herpes simplex viruses

2020 ◽  
Vol 5 (50) ◽  
pp. eaax2454
Author(s):  
Clare Burn Aschner ◽  
Lip Nam Loh ◽  
Benjamin Galen ◽  
Isabel Delwel ◽  
Rohit K. Jangra ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Immune Serum ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Wild Type ◽  
Herpes Simplex Viruses ◽  
Vectored Vaccine

Herpes simplex virus (HSV) glycoprotein D (gD) not only is required for virus entry and cell-to-cell spread but also binds the host immunomodulatory molecule, HVEM, blocking interactions with its ligands. Natural infection primarily elicits neutralizing antibodies targeting gD, but subunit protein vaccines designed to induce this response have failed clinically. In contrast, preclinical studies demonstrate that an HSV-2 single-cycle strain deleted in gD, ΔgD-2, induces primarily non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC). These studies were designed to test the hypothesis that gD interferes with ADCC through engagement of HVEM. Immunization of Hvem−/− mice with ΔgD-2 resulted in significant reduction in HSV-specific IgG2 antibodies, the subclass associated with FcγR activation and ADCC, compared with wild-type controls. This translated into a parallel reduction in active and passive vaccine protection. A similar decrease in ADCC titers was observed in Hvem−/− mice vaccinated with an alternative HSV vaccine candidate (dl5-29) or an unrelated vesicular stomatitis virus–vectored vaccine. Unexpectedly, not only did passive transfer of immune serum from ΔgD-2–vaccinated Hvem−/− mice fail to protect wild-type mice but transfer of immune serum from ΔgD-2–vaccinated wild-type mice failed to protect Hvem−/− mice. Immune cells isolated from Hvem−/− mice were impaired in FcγR activation, and, conversely, addition of gD protein or anti-HVEM antibodies to in vitro murine or human FcγR activation assays inhibited the response. These findings uncover a previously unrecognized role for HVEM signaling in generating and mediating ADCC and an additional HSV immune evasion strategy.

scholarly journals Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

2003 ◽  
Vol 77 (8) ◽  
pp. 4881-4887 ◽  
Author(s):  
Daniela Hüser ◽  
Stefan Weger ◽  
Regine Heilbronn
Keyword(s):  
Human Chromosome ◽  
Helper Virus ◽  
Productive Infection ◽  
Wild Type ◽  
Chromosome 19 ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Human Chromosome 19

ABSTRACT Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.

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