How complement shapes AAV immunogenicity beyond antibodies and T cells

For decades, adeno-associated virus (AAV) immunogenicity assessments have focused primarily on neutralizing antibodies (NAbs) and T-cell responses, and for good reason. These parameters provide solid information about the immune system’s response to AAV vectors used in many gene therapy treatments. But as therapeutic vector doses continue to increase and safety concerns intensify, researchers question whether assessing the adaptive immune response alone captures the full picture. Once on the sidelines of the discussion, the complement system is now increasingly recognized as a contributor to the serious and sometimes fatal adverse events reported in systemic AAV programs.

AAV gene therapy’s promise depends on balancing efficacy and immune risk

AAVs have gained immense popularity in gene therapy because of their ability to deliver therapeutic genetic material into target cells, enabling the treatment, and even the cure, of previously untreatable genetic diseases.

Among AAVs’ most attractive properties for clinical research are their non-pathogenic nature and serotype-based tropism. With more than a dozen naturally occurring serotypes, therapies can target specific cell types relatively safely because different AAV serotypes preferentially bind to distinct cell-surface receptors.

Nine AAV-based therapies have already been authorized in the U.S. or the EU, hundreds of clinical trials are underway, and the pace of approvals continues to accelerate. 

1. EU authorization expired in 2017 and was not renewed. 
2. Commercially discontinued by BioMarin in 2026 (sales ended). 
3. The European Commission refused marketing authorization in September 2025 after EMA opinion. 
4. EU marketing authorization was withdrawn in May 2025 for commercial reasons. 

This streak of approvals shows the great potential of AAV-based gene therapies. Yet, challenges with this technology remain. Researchers have not yet fully succeeded in controlling the body’s immune response against these vectors, facing new challenges as the doses increase.

Recent findings suggest that complement activation may play a larger role in AAV-associated toxicity than previously appreciated, prompting renewed interest in understanding when complement is activated, how it contributes to adverse events, and how these responses can be monitored and managed.

The immune response to AAV is more complex than it appears

Much of the clinical success of these non-pathogenic viruses hinges on their relatively low immunogenicity compared with other viral vectors. But despite this limited immunogenicity, immune activation against AAV capsid antigens or transgene products remains a principal obstacle to the AAV therapies’ clinical success—if not the main one—compromising both the efficacy and safety of these treatments.

Even with their encouraging safety profile, AAVs trigger immune responses that ultimately compromise therapeutic efficacy and cause serious and even life-threatening adverse events. For drug developers, potential immunogenic effects impact candidate selection, dosing strategies, patient eligibility, patient safety, and clinical outcomes, sometimes detrimentally.

Especially as clinical programs have progressively increased systemic dosing—often exceeding 1×1016 vector genomes per kg—several trials have reported serious immune‑mediated adverse events not observed at lower doses, including some reported deaths. Programs have been placed on clinical hold following these events.

As a result, regulatory agencies, such as the FDA and EMA, are increasingly scrutinizing immunogenicity assessments of gene therapies and require sponsors to characterize AAV-mediated immune responses comprehensively.

Considering the research community’s vast knowledge about AAV-triggered adaptive immune activation, what’s missing?

In healthy tissue, complement activity is tightly controlled by regulatory proteins, such as CD46, CD55, and CD59, and by soluble regulators, such as factor H and C4b-binding protein. These proteins protect the host cells from complement-mediated damage. But when the regulatory system is overwhelmed, for example, following administration of high doses of systemic AAV vectors, complement activation can become damaging rather than protective. Growing evidence suggests that both the classical and alternative pathways can contribute to AAV-triggered complement responses.

The antibody-dependent classical pathway can be activated by pre-existing antibodies developed through prior exposure to wild-type AAV or previous gene therapy, as well as by antibodies induced after infusion. In patients without pre-existing anti-AAV-capsid antibodies, IgM antibodies are typically detected 1–2 weeks after infusion, followed by IgG antibodies in the weeks after that.

But complement activation may occur before detectable antibody responses. In several studies, complement markers peaked 3–4 days after vector infusion—that is, before peak antibody response—coinciding with reduced platelet counts and elevated AST and ALT levels, suggesting liver injury or inflammation.

Although the significance of complement in AAV-associated immunogenicity has only recently become widely appreciated, clinical trials have already reported complement activation following AAV gene therapy. These observations span multiple indications, serotypes, and vector designs, suggesting the phenomenon is not limited to a single therapeutic context.

Approaching the complex biology of AAV immunogenicity

Researchers and drug developers rely on various assays to characterize AAV immunogenicity, ranging from humoral to cellular responses. 

 ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked immunospot; NAb, neutralizing antibody; TAb, total antibody. 

The research community’s accumulating understanding of AAV immunogenicity points to a more diverse and complex biology than an antibody- or T-cell-focused model alone could capture. This emerging experience ultimately highlights the need to assess that complexity more completely.

The silver lining for AAV developers lies in the capsid-dependent nature of these immune responses. Two capsid candidates that seem equivalent by antibody and T-cell assessments may differ in their complement reactivity. Identifying a complement-activating capsid in the laboratory, before it surfaces in a Phase I trial, can save candidates from ending up in clinical holds.

Test for complement activation

One way to gain more insight into the complex immune biology associated with AAV gene therapies is to monitor complement responses in patients following vector administration. Researchers have proposed measuring complement activation products in plasma, such as C4a for the classical pathway, and Ba and Bb for the alternative pathway. Shifts in these markers can indicate which complement cascade pathway is being engaged.

Still, monitoring patient outcomes requires access to patient samples and dedicated assessment tools, which limits its execution in many laboratories. Researchers and developers can instead rely on approaches that bypass the need for clinical samples.

Laboratories use ELISA-based complement activation assays on AAV-coated plates to assess complement-mediated immunogenicity in vitro. This format also provides a direct side-by-side comparison of capsid candidates, without requiring clinical samples. For example, the deposition of the terminal complement complex (TCC) reflects the complete activation of the complement cascade, from start to finish. These readouts at the very end of the cascade reveal designs that associate with cell damage and inflammatory responses.

As more patients enter clinical trials evaluating AAV gene therapies, the field will sit on ever more data that define and map new immune-response relationships. Today’s regulators and researchers recognize that complement is a necessary parameter to consider when developing AAV candidates and assessing their immunogenicity. Incorporating complement assays into the candidate pipeline can reveal meaningful differences between capsids that otherwise look equivalent by adaptive immune criteria.

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