Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models

Cardiovascular Research Center, Division of Cardiology, Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA.
Molecular Therapy (Impact Factor: 6.23). 09/2011; 20(1):73-83. DOI: 10.1038/mt.2011.177
Source: PubMed


Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models.

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Available from: Dennis Ladage, Nov 28, 2014
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    • "A limitation of this study is that the NAb analysis was performed in the same dog breed and colony and no confirmation of AAV6-specific antibodies by ELISA or Western blot was performed. Rapti et al. (28), using mainly pooled sera also found high levels of NAbs to AAV6. In this study, IgG was purified from pooled sera and blocking of AAV transduction was demonstrated, although at much lower titer than whole serum, suggesting that some other factors may play a role in AAV neutralization. "
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    ABSTRACT: Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy.
    Frontiers in Immunology 10/2013; 4:341. DOI:10.3389/fimmu.2013.00341
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    • "However, many of the serotypes of AAV commonly employed in gene therapy procedures ubiquitously infect humans, generating pre-existing immunity against the AAV capsid proteins that precludes efficient transduction following intravascular administration, and/or induces CTL responses to the transduced target tissue [14-21]. While newborn dogs have been reported to exhibit pronounced selective immunity to AAV6 [22], most animals, with the exception of some non-human primates [19,23] and, perhaps the pig [24], do not appear to harbor very robust pre-existing immunity/endogenous antibodies to many of the serotypes of AAV commonly employed as gene delivery vectors. This lack of pre-existing immunity to AAV could explain, in part, why many highly successful studies conducted in a variety of animal models have not translated into clinical success when similar approaches have been applied to human patients [25,26]. "
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    ABSTRACT: AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity.
    PLoS ONE 09/2013; 8(9):e75142. DOI:10.1371/journal.pone.0075142 · 3.23 Impact Factor
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    • "As one significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans, animal studies have demonstrated that prevention effects of in vivo AAV transduction of neutralizing antibodies are serotype specific and in a dose dependent manner [21], [23]. Rapti et al. also showed that low levels of neutralizing antibody prevented in vivo transduction by AAV9 in rats [24]. In a recent study, an intracerebral injection of AAV9-human aromatic L-amino acid decarboxylase (hAADC) has been shown to transduce antigen presenting cells (APCs) in the brain and to provoke a full immune response [17]. "
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    ABSTRACT: We have recently demonstrated that adeno-associated virus serotype 9 (AAV9)-mediated human erythropoietin (hEPO) gene delivery into the brain protects dopaminergic (DA) neurons in the substantia nigra in a rat model of Parkinson's disease. In the present study, we examined whether pre-exposure to AAV9-hEPO vectors with an intramuscular or intrastriatal injection would reduce AAV9-mediated hEPO transduction in rat brain. We first characterized transgene expression and immune responses against AAV9-hEPO vectors in rat striatum at 4 days, 3 weeks and 6 months, and with doses ranging from 10(11) to 10(13) viral genomes. To sensitize immune system, rats received an injection of AAV9-hEPO into either the muscle or the left striatum, and then sequentially an injection of AAV9-hEPO into the right striatum 3 weeks later. We observed that transgene expression exhibited in a time course and dose dependent manner, and inflammatory and immune responses displayed in a time course manner. Intramuscular, but not intrastriatal injections of AAV9-hEPO resulted in reduced levels of hEPO transduction and increased levels of the major histocompatibility complex (MHC) class I and class II antigen expression in the striatum following AAV9-hEPO re-administration. There were infiltration of the cluster of differentiation 4 (CD4)-and CD8-lymphacytes, and accumulation of activated microglial cells and astrocytes in the virally injected striatum. In addition, the sera from the rats with intramuscular injections of AAV9-hEPO contained greater levels of antibodies against both AAV9 capsid protein and hEPO protein than the other treatment groups. hEPO gene expression was negatively correlated with the levels of circulating antibodies against AAV9 capsid protein. Intramuscular and intrastriatal re-administration of AAV9-hEPO led to increased numbers of red blood cells in peripheral blood. Our results suggest that pre-immunization with an intramuscular injection can lead to the reduction of transgene expression in the striatal re-administration.
    PLoS ONE 05/2013; 8(5):e63876. DOI:10.1371/journal.pone.0063876 · 3.23 Impact Factor
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