Cell-Based Measurement of Neutralizing Antibodies Against Adeno-Associated Virus (AAV)

Jungmann, Andreas; Müller, Oliver; Rapti, Kleopatra

doi:10.1007/978-1-4939-6588-5_7

Cited by 12 publications

(5 citation statements)

References 30 publications

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“…There are two major in vitro methodologies, i.e., cell-based assays and ELISAs (enzyme-linked immunosorbent assay), which are used for screening of anti-AAV nAbs and each exhibiting distinctive advantages (123)(124)(125). Cell-based assays are more widely used as they are robust and fast.…”

Section: Humoral Immune Responsesmentioning

confidence: 99%

“…Transduction efficiency is determined at a distinct time point and expressed as percent to a no-serum/ plasma control. The titer is determined as the first dilution at which inhibition exceeds 50% (123,124,129,130), which makes this assay similar to a half-maximal inhibitory concentration (IC 50 ) assay. These assays have been performed in numerous variations, using different transgenes (green fluorescent protein (GFP), LacZ and luciferase), cell types (HEK293T or Huh7), serum or plasma, heat-inactivation or not, with adenovirus superinfection or not etc.…”

Section: Humoral Immune Responsesmentioning

confidence: 99%

“…The sensitivity of the assay was found to decrease with lower cell densities or with GFP [reviewed in (123,131)]. Of these options, luciferase and HEK293T cells are the most widely used (123,124,131), although a need for optimization remains (132). It should also be noted that different AAV purification methods produce different full/empty capsid ratios, which could also impact data transferability efforts (133).…”

Section: Humoral Immune Responsesmentioning

confidence: 99%

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Adeno-Associated Viruses (AAV) and Host Immunity – A Race Between the Hare and the Hedgehog

Rapti

Grimm

2021

Front. Immunol.

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Adeno-associated viruses (AAV) have emerged as the lead vector in clinical trials and form the basis for several approved gene therapies for human diseases, mainly owing to their ability to sustain robust and long-term in vivo transgene expression, their amenability to genetic engineering of cargo and capsid, as well as their moderate toxicity and immunogenicity. Still, recent reports of fatalities in a clinical trial for a neuromuscular disease, although linked to an exceptionally high vector dose, have raised new caution about the safety of recombinant AAVs. Moreover, concerns linger about the presence of pre-existing anti-AAV antibodies in the human population, which precludes a significant percentage of patients from receiving, and benefitting from, AAV gene therapies. These concerns are exacerbated by observations of cellular immune responses and other adverse events, including detrimental off-target transgene expression in dorsal root ganglia. Here, we provide an update on our knowledge of the immunological and molecular race between AAV (the “hedgehog”) and its human host (the “hare”), together with a compendium of state-of-the-art technologies which provide an advantage to AAV and which, thus, promise safer and more broadly applicable AAV gene therapies in the future.

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Section: Humoral Immune Responsesmentioning

confidence: 99%

Section: Humoral Immune Responsesmentioning

confidence: 99%

Section: Humoral Immune Responsesmentioning

confidence: 99%

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Adeno-Associated Viruses (AAV) and Host Immunity – A Race Between the Hare and the Hedgehog

Rapti

Grimm

2021

Front. Immunol.

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“…However, natural exposure to wild-type AAV serotypes promotes the production of specific antibodies against the AAV capsid, including a sub-class known as neutralizing antibodies (NAb) [ 3 ]. High levels of circulating NAb prevent the entry of AAV vectors into the target cell, decrease transduction efficiency and therefore decrease the expression of the therapeutic transgenes.…”

Section: Introductionmentioning

confidence: 99%

Prevalence of Neutralizing Antibodies against Adeno-Associated Virus Serotypes 1, 2, and 9 in Non-Injected Latin American Patients with Heart Failure—ANVIAS Study

Sierra-Delgado

Likhite

Bautista-Niño

et al. 2023

IJMS

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Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55–75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.

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“…Although the assay itself is easy to set up and the amount of IgG with affinity to AAV capsids can be measured accordingly, this assay does not necessarily solely represent the neutralizing activity in the sample, since non-NAbs are equally measured and reported. Cellular transduction-based in vitro assays have also been developed and widely adopted for assessing anti-AAV NAb 12, 13, 14. These assays utilize AAV vector carrying a reporter gene.…”

Section: Introductionmentioning

confidence: 99%

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay

Guo

Zhang

Chrzanowski

et al. 2019

Molecular Therapy - Methods & Clinical Development

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Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.

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Cell-Based Measurement of Neutralizing Antibodies Against Adeno-Associated Virus (AAV)

Cited by 12 publications

References 30 publications

Adeno-Associated Viruses (AAV) and Host Immunity – A Race Between the Hare and the Hedgehog

Adeno-Associated Viruses (AAV) and Host Immunity – A Race Between the Hare and the Hedgehog

Prevalence of Neutralizing Antibodies against Adeno-Associated Virus Serotypes 1, 2, and 9 in Non-Injected Latin American Patients with Heart Failure—ANVIAS Study

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay

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