#### **FIGURE 5 |TG responder status in relation with humoral and**

**systemicT-cell response against AAV**. The table below provides an overview of the individual patient cellular and humoral immune responses in relation with the clinical end point (fasting) total plasma TG. A T-cell response to the antigen was reported transient positive (transient) when

at least two consecutive sampling time points were measured positive in the ELISpot assay. The patient reported "sporadic" present recurrent non-consecutive T-cell response over time. When none, or only one sampling time was reported positive, the T-cell response was reported negative (−).

As discussed, none of these immune responses raised specific safety concerns. However, they triggered more a concern about the efficacy of the product; especially the development of any AAV-specific T-cells (48) that were thought at the time to possibly hamper the expression of LPL. A similar T-cell response was observed in gene therapy trial for hemophilia B, in which an AAV2 vector was used to deliver the human coagulation factor IX (24, 41, 42). In this trial, two patients developed a T-cell response to AAV2 capsid proteins, which was not predicted from pre-clinical studies. In those two patients, transgene expression declined subsequently to pre-treatment levels. Based on these observations, it was concluded by the investigators that a cytotoxic T-lymphocyte response to the capsid may have contributed to a loss of transgene-expressing cells.

These discussions heavily influenced the decision to include an immunosuppressant regimen in the clinical study protocols for CT-AMT-011-01 as well as CT-AMT-011-02.

As a result of these discussions, 17 patients (12 in CT-AMT-011- 01 and 5 in CT-AMT-011-02) treated with alipogene tiparvovec received a concomitant administration of immunosuppressants. Treatment-emergent anti-AAV1 antibody responses were observed in all the patients and were not affected by the immunosuppressants, neither during the time of administration nor after cessation of the administration. The AAV1–LPLS447X vector used cannot induce expression of viral proteins in host cells. Hence, AAV1 capsid proteins are expected to be only transiently presented to the immune system of the recipient after injection of AAV1– LPLS447X (3, 19, 53). Therefore, the immune suppressants drugs were given for a period of 12 weeks. A 12-week-period was considered to be sufficient for prevention of capsid immunogenicity, based on observations in monkeys (19) and the investigators early observations in humans that indicated TG levels started to rise after an initial decrease usually at some time between 4 and 12 weeks post-dosing. A combination of cyclosporine and MMF was chosen because this combination is widely used to prevent cytotoxic T-cell responses in transplant rejection. The doses of the immune suppressants proposed to co-administer with alipogene tiparvovec were according to approved doses for transplant rejection.

In study CT-AMT-011-01, humoral and cellular immune responses against AAV1 capsid proteins were observed. These responses were similar to those observed in study CT-AMT-010 although in study CT-AMT-011-01, a higher dose of AAV1– LPLS447X was used (**Figure 4**). Therefore, the immunosuppressant regimen was further optimized in study CT-AMT-011-02 by starting administration of cyclosporine and MMF administration earlier before alipogene tiparvovec administration, and by adding a bolus injection of methylprednisolone at the time of alipogene tiparvovec administration. However, in study CT-AMT-011-02, comparable humoral and cellular immune responses against AAV1 capsid proteins were observed as in study CT-AMT-011-01 (**Figure 4**).

The effect of immunosuppressants on LPL-expression and immune responses upon administration of alipogene tiparvovec is summarized in **Figure 6**. In the clinical development of alipogene tiparvovec, no untoward side effects were observed that could be assigned to the use of prednisolone or one of the other immunosuppressants.

#### **CLINICAL DATA**

#### **CHEMISTRY AND HEMATOLOGICAL VALUES**

Patients were monitored for up to 12 weeks post alipogene tiparvovec administration regarding routine hematology and biochemistry including CPK and CRP, increases of which are expected in case of local inflammatory damage at the injection site. In addition, other inflammatory markers such as neutrophil counts were also determined at several pre- and post-exposure occasions in the patients. None of the patients had neutrophil counts outside the normal range. A per-patient summary of the CRP and CPK data is given in **Figure 7**. The majority of the patients had normal CRP and CPK levels pre- and post-exposure to alipogene tiparvovec, suggesting that inflammatory reactions in the injected muscle were mild, if present, and of little clinical significance.

#### **ADVERSE EVENTS**

Alipogene tiparvovec was administered via a one-time set of intramuscular injections in the lower limbs. No consistent change in any laboratory parameter, linked to the administered product, was observed, including CPK. Injections were well tolerated with mild– moderate local injection site reactions lasting for a few days relating to the injection sites, and no change in muscle function. None of the patients showed clinical signs of persistent local inflammation at injection sites such as redness, swelling, warmth, pain, or dysfunction, upon administration of alipogene tiparvovec. There are to date no reports of muscle dysfunction in LPLD patients administered with alipogene tiparvovec.

The adverse reactions observed during the clinical development of alipogene tiparvovec are summarized in Figure S1 in Supplementary Material. Most of the adverse reactions were related to the administration procedure. All were of transient nature and resolved within days after the administration of the product. Only one serious adverse event involving muscle was seen, that was considered to be at least possibly related to alipogene tiparvovec by the Investigator (#01-002 in CT-AMT-011-02). In this subject, at 15 weeks post-administration, a transient rise in CPK, accompanied by a rise in CRP (**Figure 7**), was correlated with a low positive AAV cellular response associated with no LPL-related cellular or humoral significant response was seen in a complex of clinical symptoms and signs indicated as "polyarthralgia of imprecise origin." The subject show sporadic systemic T-cell responses against AAV across the observation period at weeks 2, 8, 14, 39. Despite those T-cell responses, the muscle biopsy of this subject which was taken 30 weeks post alipogene tiparvovec showed robust LPL-expression. In addition, the subject did not show any anti-LPL cellular or humoral response (50). No adverse effect was observed that could have been related to the immune responses discussed in the previous paragraphs.

From the clinical data obtained across studies, we conclude that there is no clinical untoward impact of the delivery of alipogene tiparvovec.

#### **DISCUSSION**

To evaluate the immunogenicity of alipogene tiparvovec, an extensive testing program was performed that included the analyses of antibody and T-cell responses to LPL as well as to AAV1. Antibody and cellular responses were measured pre-exposure to alipogene tiparvovec, and at various occasions post-exposure. In addition, biopsies were taken from the injected muscle and non-injected muscle as control, to evaluate local immune and inflammatory processes. Finally, CPK and CRP levels and neutrophil counts were measured, and patients were clinically evaluated for local and systemic symptoms indicative for inflammatory or immune reactions. Expression of LPL in the injected muscle as well as the improvement of post-prandial chylomicrons clearance in plasma was used as biochemical markers for efficacy.

The success of *in vivo* gene therapy not only depends on the ability to control the immune response toward the vector, but

**FIGURE 7 | A per-patient summary of CRP and CPK values pre- and post-exposure to alipogene tiparvovec**. Scheduled visits pre-treatment and post-treatment have been included. The post-treatment visits were at day 1 and weeks 1, 2, 4, 8, and 12 for studies CT-AMT-010 and CT-AMT-011-01. Weeks 14, 26, 39, and 52 were added for study CT-AMT-011-02. In the study

also to monitor any potential reaction to the therapeutic protein expressed from the transgene. The data obtained from patients with LPL-deficiency who received a single treatment with multiple injections of alipogene tiparvovec, support the initial expectation that the protein product is minimally immunogenic, if at all: neither treatment-emergent antibody responses against LPL nor T-cells responses against LPL were found. Thus, the expressed protein itself appears to be non-immunogenic.

Concerning anti-AAV immunity, the majority of healthy individuals are exposed to AAV during lifetime (2, 54). Hence it is not surprising that 15 of 27 patients had pre-existing antibodies against AAV1. There was no difference among patients with or without these antibodies with regard to detectable LPL-expression in the biopsies or improvement of post-prandial chylomicron clearance, suggesting that pre-existing anti-AAV1 antibodies did not appear to prevent LPL transgene expression and clinical efficacy. Therefore, pre-existing anti-AAV1 antibodies likely have no effect on the efficacy of alipogene tiparvovec following intramuscular administration. All patients showed treatment-emergent antibody responses to AAV1 and there was no difference in anti-AAV1 antibody response between the various dosing cohorts. AMT-011-01, the isolated elevation of CRP for subject 014 was concomitant with a transient medical condition. In the study AMT-011-02, the isolated elevation of CRP for subject 01-001 was concomitant with a transient medical condition. The isolated elevation of CRP and CPK for subject 01-002 was related to the general clinical condition of the subject.

The markers that were used to demonstrate LPL-expression and functionality after the delivery of alipogene tiparvovec are complex. Therefore, the present report is focusing on LPLexpression in muscle biopsies and post-prandial chylomicron clearance as marker for systemic LPL activity. For the direct measurement of LPL-expression, we made use of biopsies that were obtained at different time points up to 12 months after drug administration. However, and as described above, not all patients gave their consent to this procedure which can present some variability in the execution. Nineteen patients provided their consent and in total 20 biopsies were obtained, which all were examined for LPL-expression. In 12 of the 20 biopsies, LPLexpression was found irrespective of the presence of antibodies against AAV1. The second biochemical marker for successful gene delivery in this review is the level of clearance of post-prandial chylomicrons in plasma, which reflect the systemic activity of LPL (49). This marker was however, included only in the study CT-AMT-011-02. The post-prandial chylomicrons plasma levels were shown to be significantly reduced in all five patients included in this study, independently of the presence of anti-AAV1 antibodies.

Altogether, these observations demonstrate that anti-AAV1 antibody responses did not exclude sustained transgene expression nor did impair the systemic biological activity of the expressed protein. Our results further support that treatment-emergent anti-AAV1 antibody responses do not necessarily have any influence on the long-term efficacy and safety of AAV-based gene therapy.

Treatment-emergent T-cell responses against AAV1 capsids were measured with the ELISpot assay and were observed in 18 of the 27 patients. However, it has to be noted that the patients treated with the higher dose, 1 × 1012 gc/kg, were more prompt to develop an AAV1-specific T-cells responses. Somewhat higher T-cell responses were noticed in some patients upon cessation of the immunosuppressants, pointing to a suppressive effect on the T-cell responses. It is therefore not possible to make definitive conclusions regarding the effect of immunosuppressants on T-cell responses to AAV1. The immunosuppressants did not affect antibody levels as was expected since the regimen was mainly aiming at reducing potential T-cell responses. MMF and cyclosporine are both described to be effective in suppressing cytotoxic T-cell responses. MMF has an effect on B-cell proliferation, because it inhibits *de novo* guanosine nucleotide synthesis, a pathway commonly required for both T- and B-cell proliferation. Cyclosporine specifically inhibits T-cell activation by inhibiting IL-2 production and exerts limited, if any, effect on B-cell proliferation. Thus, the increased anti-AAV1 titers are not surprising. Similar observations were made in a study in monkeys (52) when administrating AAV8 hFIX together with immunosuppressants consisting of MMF and tacrolimus. In the third study of the three interventional studies for alipogene tiparvovec, CT-AMT-011-02, prednisolone was administered in the form of a bolus injection to prevent the release of substances that mediate inflammation and to enhance the potency of the other immunosuppressants used.

Our studies should be considered in the context of the growing body of clinical and pre-clinical studies evaluating the role of capsid-specific T-cells in AAV gene. The immunogenicity data found in the clinical studies conducted with AAV-based vectors in human, show that immune responses against AAV capsid proteins can vary widely and amongst others are influenced by the target organ, route of delivery, and dosing schedule. When targeting the muscle in humans, T-cell responses directed to the capsid antigen were documented in AAT-deficient subjects receiving intra-muscular injection of an AAV1–AAT vector (25, 55), in study on AAV1-α-sarcoglycan in limb-girdle muscular dystrophy subjects (35) and in our own LPL-deficient subjects. The controversial aspect of the capsid-specific T-cell hypothesis is whether the vector sensitizes transduced cells to become targets for CTLmediated clearance by virtue of MHC presentation of peptides from the input capsid protein. Also, immune modulation was used which could have impacted on the AAV-specific immune responses, our study provides the most direct and extensive test of this hypothesis because we observed transgene expression until 52 weeks (long-term follow-up) after injection of AAV1–LPLS447X in the muscle, despite the detection of circulating T-cell specific for AAV capsid peptides in some subjects and persistent focal infiltrates in all subjects for whom transgene expression was detected. These data clearly demonstrate that transgene expression can persist, despite the presence of capsid-specific T-cells and cellular infiltrates. Sustained transgene expression in the presence of T-lymphocyte responses have been reported in the literature in experimental animals and in different tissues (56, 57) and humans (25, 35, 37). However, whereas attention has been focused initially on the AAV capsid as target of an undesirable T-cell response (24, 41, 42), observations made by the groups of Dr C. Walker (58) and supported by observations of others (59) suggest that loss of function and programed death by most tissue-infiltrating AAV-primed T-cells seem to argue against their direct participation in clearance of AAV-vector-transduced target cells. It has been described that T-cells in AAV-vectors related infiltrates present characteristics of anergy (55). Such T-cell infiltrates are therefore generally considered as unable to initiate cellular self-destruction and therefore do not impact on efficacy of transgene expression.

Another mechanism via which T-cells may affect LPLexpression is stimulating the proliferation and differentiation of B cells that subsequently form antibodies against AAV1. However, as is discussed above, anti-AAV1 antibodies seem to have no impact on LPL-expression and there is no evidence whether antibody-dependent cell-mediated cytotoxicity played a role. As mentioned before, a sustained long-term transgene expression was observed after intra-muscular injection, despite the presence of circulating antibodies directed against the AAV1 capsid peptides. The results obtained with alipogene tiparvovec demonstrate that the presence of the anti-AAV1 humoral immune response had no apparent influence on the long-term efficacy of the therapy. The same observation has been reported in other clinical studies (25, 35, 37, 47, 55).

Multiple intra-muscular injections of the vector and supposed inflammatory and immune reactions ensuing at the injection sites raise concerns about inflammatory damage in the injected muscle. However, except for transient mild local procedural symptoms at the injection sites, no clinical symptoms such as swelling, pain, or dysfunction pointing at inflammatory damage were observed in the patients. In addition, serial monitoring of CRP and CPK revealed normal levels of these markers in most patients. Occasional elevations of CRP and CPK were seen in two and in one patient, respectively, without any clinical correlation. In addition, though a mild mononuclear infiltrate was observed in 14 of the 19 patients of whom a biopsy was obtained, this infiltrate lacked substantial cytotoxic T-cells activity. Hence, no clinical, biochemical, histochemical, or immunological evidence for inflammatory muscle damage at the injections sites was found.

All together, the data collected on systemic and local immune responses induced by intra-muscular injection of alipogene tiparvovec demonstrate the absence of impact on safety and did not compromise LPL transgene expression. These findings indicate that muscle-directed AAV-based gene therapy through the intravascular route remains a promising approach for the treatment of human diseases.

#### **REGULATORY PERSPECTIVE**

During the assessment of the Glybera marketing authorization dossier, the fact that no responses had been seen against the expressed LPL protein was considered as a positive safety asset and no material concerns were expressed.

The necessity of using immunosuppressants was not proven during the clinical development of Glybera since the regimen seemed not to improve efficacy whilst having a major negative impact on the safety aspects. It remains questionable whether it makes clinical sense to co-administer immunosuppressants with any AAV-based vector. However, in the case of Glybera, since LPLD is such a rare disease, it will not be possible to further assess long-term clinical efficacy in absence of any immunosuppressants within the present indication.

During the regulatory review by the European Medicines Agency, multiple questions arose on whether the cellular responses to the viral capsid proteins could have any meaningful negative effect on the long-term safety or efficacy of alipogene tiparvovec. Our data clearly demonstrate that transgene expression can persist, despite the presence of capsid-specific T-cells and cellular infiltrates and without apparent toxicity or attenuation of transgene expression. Furthermore, the purity of the vector preparations (in terms of total amount of viral proteins injected versus dose in genome copies) and impurities profile of the vectors used in the various clinical trials described in the literature may be very divergent and therefore may lead to very different immune responses. Nonetheless, the scientific debate has been powerfully influenced by previous findings with other vectors and by the hypothesis that the vector sensitizes transduced cells to become targets for CTLmediated clearance. Therefore, since the safety data on alipogene tiparvovec have been collected in a small number of patients, their clinical relevance and possible interpretations were considered not fully unequivocal and further data collection has been requested by the European Medicine Agency post-approval of Glybera®. Such data will be collected from all treated patients in future in a LPLD registry, thus allowing for long-term data analysis.

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00082/ abstract

#### **REFERENCES**

for stable liver transduction in vivo. *J Virol* (2001) **75**:6969–76. doi:10.1128/JVI. 75.15.6969-6976.2001

**Conflict of Interest Statement:** The funding body (uniQure) was involved in all aspects of the study. The authors (Valerie Ferreira, Florence Salmon, and Harald Petry) are employes of uniQure.

*Received: 09 September 2013; accepted: 16 February 2014; published online: 03 March 2014.*

*Citation: Ferreira V, Petry H and Salmon F (2014) Immune responses to AAVvectors, the Glybera example from bench to bedside. Front. Immunol. 5:82. doi: 10.3389/fimmu.2014.00082*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Ferreira, Petry and Salmon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Immunological monitoring to rationally guide AAV gene therapy

### **Cedrik Michael Britten1,2\*, SteffenWalter 3 and Sylvia Janetzki 4**

1 Translational Oncology, University Medical Center, Johannes Gutenberg-University Mainz (TRON gGmbH), Mainz, Germany

2 Association for Cancer Immunotherapy (CIMT), Mainz, Germany

3 Immatics Biotechnologies GmbH, Tübingen, Germany

4 ZellNet Inc., Fort Lee, NJ, USA

#### **Edited by:**

Federico Mingozzi, Université Pierre et Marie Curie/Genethon, France

#### **Reviewed by:**

Valerie Ferreira, uniQure, Netherlands Oumeya Adjali, Institut National de la Santé et de la Recherche Médicale, France

#### **\*Correspondence:**

Cedrik Michael Britten, Translational Oncology (TRON), University Medical Center, Johannes Gutenberg-University Mainz gGmbH, Langenbeckstr. 1, 55131 Mainz, Germany

e-mail<EMAIL_ADDRESS>
Recent successes with adeno-associated virus (AAV)-based gene therapies fuel the hope for new treatments for hereditary diseases. Pre-existing as well as therapy-induced immune responses against both AAV and the encoded transgenes have been described and may impact on safety and efficacy of gene therapy approaches. Consequently, monitoring of vector- and transgene-specific immunity is mandated and may rationally guide clinical development. Next to the humoral immune response, the cellular response is central in our understanding of the host reaction in gene therapy. But in contrast to the monitoring of antibodies, which has matured over many decades, sensitive and robust monitoring ofT cells is a relatively new development.To make cellular immune assessments fit for purpose, investigators need to know, control and report the critical assay variables that influence the results. In addition, the quality of immune assays needs to be continuously adjusted to allow for exploratory hypothesis generation in early stages and confirmatory hypothesis validation in later stages of clinical development.The concept of immune assay harmonization which includes use of field-wide benchmarks, harmonization guidelines, and external quality control can support the context-specific evolution of immune assays. Multi-center studies pose particular challenges to sample logistics and quality control of sample specimens. Cooperative groups need to define if immune assessments should be performed in one central facility, in peripheral labs or including a combination of both. Finally, engineered reference samples that contain a defined number of antigen-specific T cells may become broadly applicable tools to control assay performance over time or across institutions.

**Keywords: immunological monitoring, assay harmonization, gene therapy, adeno-associated virus, biomarkers**

#### **INTRODUCTION**

Inherited diseases account for a substantial number of hospital admissions of children but also affect adults. Despite progress in science, efficient treatments that go beyond supportive care are not available for most of these diseases. Gene replacement therapies bear a high potential to address the medical need in affected patients and have been introduced to clinical testing about 20 years ago (1). After many draw-backs in the past an increasing number of reports of successful gene therapies in patients with hemophilia (2), Leber's congential amaurosis (LCA) (3), or X-linked adrenoleukodystrophy (4) were recently published. In November 2012, Glybera® (alipogene tiparvovec) was the first human gene therapy to receive a market approval from the European Medicines Agency (EMA) suggesting that the field has reached a turning point which may give raise to additional approved gene therapies in the future (5).

Most strategies applied for gene replacement focus on two types of vectors to reach stable transfer of functional gene products into selected target tissues, namely lentiviral vectors for *ex vivo* gene transfer into hematopoietic cells or other stem cells, or adenoassociated virus (AAV) for *in vivo* gene transfer to various target tissues such as muscle, the liver, the retina, lung, or the brain. Despite successes reported for AAV gene therapy approaches it has become clear that humoral and cellular immune responses against both the vector as well as the transgene may negatively impact on safety and efficacy of gene therapy approaches (6). Humoral responses against AAV frequently occur in the population, increase by age and are efficiently induced following a single administration of AAV-based therapies. Neutralizing antibodies against AAV have been shown to negatively influence transduction rates and may counter-act therapy approaches with systemic delivery of AAV in particular. More recently, pre-existing antigen-specific T cells recognizing AAV capsid proteins have been suggested to be an independent factor leading to reduced transduction rates on one hand and immune-mediated clearance of transduced cells expressing capsid proteins on the other hand, which led to immune-related adverse events in patients (7, 8). The first results showing that human CD8+ T cells to AAV capsid could limit the efficacy of the gene transfer originated form an initial trial of AAV gene transfer to the liver for treatment of hemophilia (7). Here it could be shown that expression of the initially expressed factor IX (F.IX) disappeared after several weeks, accompanied by a transient elevation of transaminases of the liver that were shown to be mediated by antigen-specific T cells directed against AAV but not the therapeutic F.IX that eliminated vector-transduced hepatocytes. In addition, extensive T cell monitoring from multiple studies performed in gene transfer to the muscle indicate that there is a dose dependent increase in treated patients showing T cell reactivity against the AVV capsid. Interestingly, currently available data suggest that gene transfer to immune-privileged body compartments such as the eye and central nervous system so far did not lead to detectable immune responses to the capsid or the transgene. In summary, the immunogenicity data found in the clinical studies conducted with AAV-based vectors in human and available in literature, confirm that immune responses against AAV capsid proteins can vary widely and amongst others are influenced by the target organ, route of delivery, and dosing schedule (9). Also antigenspecific T cells against the encoded therapeutic gene have been described and may reduce efficacy of gene therapy (10). All these findings explain why approaches to better understand or block immune responses following gene transfer with viral vectors have recently come into focus in the field (11).

Adeno-associated virus-specific T cells have been shown to be directed against AAV capsid proteins inducing transient hepatitis following i.v administration of AAVs targeting the liver (7). Although transient toxicity may be acceptable for regenerating tissues such as the liver in regard of safety, immune-mediated removal of AAV-transduced cells may become a major obstacle for gene transfer into toxicity relevant organs such as the brain or the heart. AAV-specific immunity may also lead to safety concerns as well as efficacy loss in replacement of the retinal pigment epithelium-specific 65-kDa protein gene in the retina in patients with LCA as re-therapy in the other eye may be mandated (12).

The currently available data on AAV-therapy induced immunity supports the notion that detailed knowledge of the presence of antigen-specific T cells prior to gene therapy as well as the occurrence of vector- and transgene-specific immunity following therapy may guide clinical decision making in the future (6, 9). Examples for proactive use of immunological data for the benefit of patients could be: (i) the selection of patients that have a high likelihood to exert the wanted effects, (ii) identification of patients that may need an adaption of the therapy (e.g., lower doses, improved vector), or (iii) receive concomitant immunesuppression until immunogenic capsid proteins are cleared. It may also be used to (iv) identify patients at risk for adverse immune reactions following second administration of the vector, or (v) be early indicators of loss of function of the encoded gene product.

In order to confirm the hypothesis that data generated by immunological monitoring can indeed impact on clinical decision making to enhance AAV-mediated gene therapy, more systematic analyses of AAV-specific T cell immunity are mandated and have even been recommend by NIH Recombinant DNA Advisory Committee (13). The fact that T cell assays may have a higher complexity and variability as compared to assays to quantify soluble analytes explains why all investigators that perform correlative biomarker studies should know, control and report on the variables of assay conduct that are known to critically affecting immune assay performance. The critical variables were identified by large scale proficiency programs conducted by the Cancer Research Institute's Cancer Immunotherapy consortium (CRI-CIC), the Cancer Immunotherapy Immunoguiding Program (CIMT-CIP) (14), and the Human Immunophenotyping Consortium (15). In addition analytical labs are advised to optimize and standardize sample logistics and assay conduct prior to testing specimens from clinical trials even at the earliest stage of clinical development in which hypothesis generation represents the primary aim of immune assay use. Assay qualification and validation become mandatory requirements when a hypothesis generated in exploratory research from early clinical development has to be confirmed in advanced clinical development (16). This gradual evolution of immune assays from first exploratory use toward validated assays performed under Good Clinical Laboratory Practice (GCLP) standards may be supported by assay harmonization at any stage (**Figure 1**).

#### **IMMUNE ASSAY HARMONIZATION AND VARIABLES THAT IMPACT ON ASSAY PERFORMANCE**

A plethora of assays exists to evaluate specific T cell responses, wanted or unwanted. These assays differ in their sensitivity to detect low frequency T cells, quantity of information obtainable, ability to determine structural and/or functional features of T cells, and their complexity in qualification and validation demands (17). For years the field of T cell monitoring has been plagued by a seemingly inherent variability in assay results, even between assays evaluating the same sample in the same laboratories or by different laboratories. A divergence of Standard Operating Procedures (SOPs), the introduction of new reagents and tools by manufacturers, preferences by the assay performing scientist, varying availability of equipment or the qualification, and training status of personnel, has further complicated the issue. Due to the lack of stringent QC procedures as required for clinical diagnostic tests, many assay "cooks" brewed their own results "soups" following their recipe without knowledge of how good or how bad that soup

**performance of immune assay in one lab**. Immune assay harmonization can support quality of immune assays at any step of assay development and use, starting from the initial assay development until assay validation.

"tasted." An additionally complicating fact is the limited availability of test samples, and the intrinsic biological variability in performance of such samples, a factor well known today that has caused a shift in considerations concerning sample handling for T cell monitoring (18–20).

This heterogeneous landscape of assays and protocols exists in cancer immunology, infectious diseases, transplantation immunology, and gene therapy. As outlined earlier a multitude of gene therapy approaches exist that (i) address several unrelated diseases, (ii) apply various viral vectors and serotypes, (iii) target different tissues (e.g., liver, muscle, retina, central nervous system), (iv) are applied using different administration routes and (v) deliver various therapeutic gene products. The fact the gene therapy comes in different flavors mandates a product specific adaption of the immunological monitoring that acknowledges therapy-related peculiarities and integrates available knowledge on immunogenicity and epitope hierarchy of both the applied vectors and therapeutic transgenes. The fact that there is not one single assay format that is applicable for all gene therapy trials poses a challenge on the gene therapy community to agree on some standards that may be used across a variety of clinical products and studies to generate results that may be comparable across institutions.

First activities examining the differences in T cell assay performance between laboratories evolved more than 10 years ago from cancer immunologists and researchers in the HIV field that initiated proficiency panel projects for ELISPOT (21, 22). This concept was soon adopted by two non-profit organizations from the cancer research field, the CIMT-CIP and CRI-CIC, and elevated to highly efficient programs that address proficiency testing of assay performance of individual labs and the identification of variables influencing the assay outcome at large (14). By sending out pretested samples in a blinded fashion to a large number of laboratories which had to test those sample with a given T cell assay following their own protocol, and report the results back to a central site including details about how they performed the assay, CRI-CIC and CIMT-CIP were able to not only give feedback on the performance of each individual laboratory in comparison to the other participating laboratories, but also identified critical protocol variables that influence the results reported back. These studies indicated that independent of SOPs applied, the results of a significant proportion of labs accumulated around an overall median value in results, while others were outliers. With the help of protocol information provided, initial recommendations for SOP adaptations were deduced and introduced to the field for the next testing round, where it could be confirmed whether these recommendation can indeed improve performance. This iterative testing process allowed the identification of protocol steps that, independent of the SOP applied, could elevate the assay performance of labs (23–26). These findings are summarized in harmonization guidelines which are now increasingly used by laboratories (**Figure 2**). The introduction of such harmonization guidelines has led to significant improvement in response detection across laboratories (27) and decreased the overall variability in results between laboratories. Further investigations of specific recommendations have led to even more detailed guidance in the harmonization process, for example by investigating the use of serum-free freezing and assay media in ELISPOT (28, 29), the implementation of a dump channel and viability marker in MHC-peptide multimer staining (26) and obtaining gating recommendations for flow-based experiments (30, 31). Such assay harmonization activities, consisting of: (i) a mechanism for regular external proficiency testing with fast feed-back loops to participants, (ii) dynamic harmonization guidelines and (iii) benchmarks for assay performance, can support investigators to improve and maintain quality of their assays at any stage of development (**Figure 1**).

While this assay harmonization process is ongoing and an increasing number of labs apply its results, there exists a discrepancy between what is being done in laboratories, and what is reported when publishing results from T cell assays. To address the lack of transparency often observed in publications that hinders the accurate interpretation and replication of results, as well as the comparison of results from different laboratories, the Minimal Information about T cell assays (MIATA) project was introduced (32). A 3-year extensive public consensus process involving more than 100 scientists from the different fields of translational immunology and regulatory agencies as well as editors from scientific journals resulted in a final reporting framework for results of T cell assays (33) that summarizes the minimal information to report about assay conduct. Easy implementation tools are available online (miataproject.org), and MIATA-compliant papers are listed with a link to the original publication.

These activities, together with available harmonization guidelines, if followed, elevate the T cell immune monitoring field to a level that allows the creation of high quality data that support immunotherapeutic developments and advances, and hence deserve the embracement by the translational science community at large.

#### **IMMUNE ASSAYS IN MULTI-CENTER CLINICAL TRIALS**

A further complicating factor for performing T cell immunomonitoring is the complexity and fragility of the analyte. In most cases, analysis is focused on T cells obtained from peripheral blood; less often (as more invasive sampling is required) T cells infiltrating other tissues such as bone marrow or skin are analyzed.

Importantly, functional T cells assays are sensitive to the time passed since the sample has been taken on a typical scale of hours (18, 19, 34). In terms of assay validation language, the sample stability is low. This can be both influenced by the inherent instability of the T cells and by the instability of the sample matrix such as granulocytes that become activated over time (35). For some assay parameters, this time window may be prolonged by adding stabilizers or by isolating the T cells without cryopreservation prior to shipping the sample (36). More generally, there are two known solutions to this logistical challenge: (i) all measurements are being performed within short time using fresh samples, or (ii) all T cells are isolated from the biological samples and cryopreserved before the assay.

The first solution may be especially attractive in the case of monocentric studies. A major drawback of such a scenario is that no retrospective analyses will be possible, longitudinal samples of a patient cannot be analyzed within the same assay, and in the case of multi-centric clinical trials this requires all immune assay parameters to be fully standardized at each site where patients are recruited. Therefore, this solution that has been termed as "peripheral analysis" (37, 38) poses high demands on assay standardization, which should be completed before a trial is initiated. Nevertheless, using fresh samples does not control inter-assay variability, and hence can introduce variability in measurements of samples obtained from different time points from a patient, which can confound the response determination between time points. Determining the precision between measurements of samples obtained from different time points is logistically challenging, hence the introduced degree of variability by testing fresh samples cannot be easily defined.

The second solution requires isolation and cryopreservation of the T cells shortly after the sample has been taken. Once the cells are frozen and kept at cryogenic temperatures, they are stable for very long time periods and can be shipped over great distances to enable batch-wise analysis within one central lab, thereby reducing assay variation. This approach does control variability by allowing the simultaneous testing of samples obtained from different time points in one assay. However, isolating and cryopreservation of T cells from biological samples is a relatively laborious process and is not performed as part of the tests for clinical routine. Therefore, if it has to be implemented within a multi-centric clinical trial, all aspects of blood collection, transport to the laboratory isolating T cells, conduct of the isolation and cryopreservation process at the lab and shipping of the frozen sample should be standardized between labs using fully comparable processes and materials (39). This solution has been termed as "central analysis" (37, 38) and poses high demands on standardized sample collection, while allowing more flexibility on the assay that are later being performed with the collected samples. Recent harmonization efforts have addressed cryopreservation challenges, and provide some guidance (28), while others are still ongoing. The performance of cell isolation and cryopreservation may be monitored by counting viable cell numbers at the time of isolation and after thawing the samples and in the case of blood samples the yield can be additionally monitored by comparing the numbers of isolated cells to the numbers of lymphocytes and monocytes from a routine hematology assessment.

To demonstrate the reproducible performance of immunoassays over time in one lab during the conduct of a clinical trial, and between labs as in the case of peripheral analysis, a stable source of reference samples that can be repeatedly analyzed is of central importance. For T cell assays, the generation of a stable reference sample is a challenging task compared to chemical assays, for example, where a small molecule can be spiked into a sample matrix at pre-defined concentrations. In the past, labs have often prepared local reference samples by aliquoting and cryopreserving cells from one large blood draw that contained T cells of a known reactivity. Such reference samples are limited in size, not comparable between different batches, and do not contain pre-defined "known" numbers of T cells. Recently, a first generation of reference samples that contain a defined number of functional antigen-specific T cells have been developed by CIP (40). The technology is based on transfer of antigen-specific T cell receptors (TCRs) into primary lymphocytes using viral gene transfer. Second generations of reference samples that are based on the use of *in vitro* translated RNA for TCR gene transfer are under development and has been shown to be simple to manufacture, robust, and sensitive, stable of at least 1 year and shown to be suitable for ELISPOT assays, HLA-peptide multimer staining as well as cytokine flow cytometry applications (unpublished data). A completed proficiency panel with 12 labs in Europe and the USA showed that the technology is easily transferable to other labs and across protocols. Given the currently available data on T cell reactivity against the AAV capsid, a set of reference samples engineered with capsid-specific TCRs for the most prevalent

HLA-restrictions may become a useful tool for the gene therapy community and help to control assay performance and increase comparability across different studies using AAV gene transfer. Thus RNA-based TCR-engineered reference samples may become a standard tool to control immune assay performance over time and help destigmatizing T cell assay use in clinical trials an become yet another component of immune assay harmonization.

#### **CONCLUSION/SUMMARY**

Systematic immune assessments in gene therapy trials using quality controlled immune assays will help to understand the immunological interactions between AAV-based therapies and the adaptive immune system. Associating immunological biomarker data with data on clinical safety and efficacy will enable the field to use

#### **REFERENCES**

immunomonitoring data for clinical decision making to improve gene therapy approaches. Prior to use of immune assays investigators need to know and control those assay variables that determine the quality of sample specimens and assay results and should provide structured reporting on the assay setup in publications and reports. By providing a fast feedback of assay performance over time or across institutions, assay harmonization can support immune assay development and use and thus may be considered as a tool to enhance biomarker research and development of new immune- and gene-therapies.

#### **ACKNOWLEDGMENTS**

Cedrik Michael Britten was supported by a grant from the Wallace Coulter Foundation (Miami, FL, USA).

MP. Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. *AIDS Res Hum Retroviruses* (2005) **21**:68–81. doi:10.1089/aid.2005.21.68

density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples. *BMC Immunol* (2006) **7**:11. doi:10.1186/1471-2172-7-11

study. *Cancer Immunol Immunother* (2013) **62**:489–501. doi:10.1007/ s00262-012-1351-0

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 June 2013; accepted: 27 August 2013; published online: 12 September 2013.*

*Citation: Britten CM, Walter S and Janetzki S (2013) Immunological monitoring to rationally guide AAV gene therapy. Front. Immunol. 4:273. doi: 10.3389/fimmu.2013.00273*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Britten, Walter and Janetzki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Pre-clinical assessment of immune responses to adeno-associated virus (AAV) vectors

#### **Etiena Basner-Tschakarjan1\*, Enoch Bijjiga2 and Ashley T. Martino2\***

1 Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA

2 Department of Pharmaceutical Sciences, St. John's University, Queens, NY, USA

#### **Edited by:**

Federico Mingozzi, UPMC Paris 6 and Genethon, France

#### **Reviewed by:**

Gloria Gonzalez-Aseguinolaza, Fundación para la Investigación Médica Aplicada, Spain Jonathan D. Finn, Arthrogen B.V., Netherlands

#### **\*Correspondence:**

Etiena Basner-Tschakarjan, Children's Hospital of Philadelphia, 3501 Civic Center Blvd, Suite 5400, Philadelphia, PA 19104, USA e-mail: basnertschakarjane@ email.chop.edu; Ashley T. Martino, St John's University, 8000 Utopia Parkway, SAH Room 141, Jamaica, NY 11437, USA e-mail<EMAIL_ADDRESS>
Transitioning to human trials from pre-clinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxicT-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity and explored methods for bypassing these responses. Many efforts toward measuring innate immunity have utilized Toll-like receptor deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigenspecific T-cells as well as cytotoxicity toward AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T-cell proliferation, but actual transgene level reduction and parameters of cytotoxicity toward transduced target cells have only been shown in one model. The model utilized adoptive transfer of capsid-specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells as well as modulating the response pharmacologically has also been explored.

**Keywords: immune responses, adeno-associated virus, pre-clinical models, AAV, inhibitory AAV**

#### **INTRODUCTION**

Over the past 20 years, a full spectrum of immune responses to AAV has been assessed to include innate responses, humoral responses, and T-cell responses. In addition to the emphasis on pre-existing antibodies to AAV, cytotoxic T-cell (CTL) responses to AAV have emerged as a challenge for clinical applications. Roughly 70% of the human population has neutralizing antibodies (NAb) titers to AAV2 that can interfere with gene therapy (1–4). The use of different AAV capsids may overcome this challenge yet lower antibody titers to alternative capsids as well as cross-reactivity of AAV2 antibodies is concerning (3, 4).

Since mice and dogs do not have pre-existing immunity to AAV, correction in coagulation actor IX (FIX) deficient animal studies have been sustained without concern of immune responses (5–7). Unfortunately, the lack of immunity in pre-clinical studies did not fully reflect clinical results. In patient studies using AAV2 or AAV8 expressing normal or self-complimentary (sc) hFIX respectively, AAV1 expressing α1-antityrpsin (AAT), and Lipoprotein Lipase (LPL) correction, capsid-specific CTL responses have emerged, particularly at high doses (8–11). In some cases, existence of capsid-specific CTLs did not significantly impact correction as

seen in AAT studies involving intramuscular (IM) injections (10). In hemophilia B (HB) liver studies, correction at 10% of normal was achieved in high dose cohorts. This level was only sustained 4– 8 weeks and a spike in liver transaminases coincided with a drop in hFIX levels (8, 9), which was coordinated by capsid-specific CTLs. With the occurrence of CTL responses and presence of pre-existing antibodies to AAV capsid there has been a concerted effort to develop suitable pre-clinical approaches for complete assessment of immune responses and to develop intervention techniques.

#### **CHARACTERIZATION OF INNATE IMMUNITY TO AAV**

Pattern recognition receptors (PRRs) on monocytes recognize AAV particles and trigger innate immunity (12–15). TLR2 and TLR9 have been identified as the PRRs for AAV. TLR2 triggers responses to the capsid while TLR9 detects the viral genome. Studies have been performed in myeloid differentiation primary response 88 (*myd88*−/−), *tlr9*−/− and *tlr2*−/− deficient mice to pinpoint TLR pathways associated with AAV recognition. Additionally, adaptive responses have been assessed in these mice. Moreover, studies were performed in interferon β receptor of KO mice (*ifnr*−/−) to establish a cytokine for developing adaptive responses (13, 14). Measuring cytokine secretion from differentiated monocytes isolated from these KO mice was used to segregate responses to AAV2. Absence of type I interferon (IFN) responses in AAV-activated plasmacytoid dendritic cells (pDCs) isolated from *tlr9*−/− mice as well as *myd88*−/− but not *trl2*−/− mice suggests that IFN responses are due to the vector genome, not the capsid (12–14).

To further assess the immune responses to AAV in KO mice, cellular and humoral responses were measured. Assessing NAbs, serum IgG2a or CTL responses to AAV capsid in mice deficient in innate immunity was used to determine a pathogen recognition link between innate and adaptive response. Both *tlr9*−/− and *myd88*−/− mice showed absence of adaptive responses to AAV (13, 14). AAV antibody titers were assessed by enzyme-linked immunosorbent assay (ELISA) while CTL responses were assessed using enzyme-linked immunosorbent spot (ELISpot) analysis of IFN-γ secretion from isolated splenocytes.

Efforts to determine if genome or capsid modifications alter innate responses have been pursued. Gene expression in liver of AAV2-infected mice showed an increase in inflammatory mediators and TLR9 transcripts, but not TLR2. Additionally, the responses remained the same between capsid variants but the sc genome resulted in a significant increase in transcription of inflammatory mediators. Determining cellular recruitment also indicated the sc-genome significantly increased inflammation. This was measured by staining liver sections for neutrophils, Mac-1+ macrophages and cd335+ natural killer cells. The use of *tlr9*−/− mice as well as TLR9 antagonists prevented innate immune responses, showed a reduction in adaptive immunity, and demonstrated a plausible connection between TLR9 and development of antibodies and CTLs (14).

Efforts to isolate immune responses to AAV input capsid have also been performed. TLR2 and TLR4 are highly associated with recognition of viral structural proteins (16). The production of empty capsids devoid of genomes and the use of HEK293 reporter cell lines, which secrete IL-8 due to activation of stably expressed TLR2 or TLR9, have aided in pinpointing PRRs associated with capsid. HEK293/TLR2 but not HEK293/TLR9 showed activation by empty vectors (15).

#### **HUMORAL IMMUNITY**

Epidemiology studies have predicted that ~70% of the population is seropositive for AAV2 (1–4). Further studies have estimated a lower degree of seroprevalence for other AAV capsids (4). Moreover, 20–40% of the population has NAb titers (1:80) against AAV2 capable of reducing transduction of Huh7 cultured cells by 50% using a Multiplicity of Infection of 104 . Additionally, the ability of AAV2 NAb to reduce transduction efficiency against alternative serotypes has developed concern for cross-reactivity.

#### **CHARACTERIZING NAb INHIBITION OF AAV**

Dot Blot studies, NAb assays and cross administration of AAV vectors in animals have been used to determine cross-reactivity of AAV antibodies. The cross-reactivity of αAAV1 or αAAV5 antibodies was tested against a dot blot containing AAV1,AAV2,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 capsid. AAV1 antibodies showed strong adherence to only AAV6, while AAV5 antibodies had

minimal adherence to AAV1 (17) and only at high concentrations of monoclonal antibodies.

In NAb *in vitro* assays, AAV vectors are pre-incubated with serum containing NAbs prior to infection of cultured cells and can be used for assessing cross-reactivity when alternative capsids are selected. Using this assay, it was determined that NAbs against AAV1 or AAV5 had limited impact on transduction from the other capsid. Additionally, engineered capsid modifications to AAV2 or a chimeric AAV capsid overcame antibody neutralization based on parental AAV antigens (4, 17–19).

Neutralizing antibody in mice also determined the potential of AAV antibodies to cross react with a different capsid using *in vivo* approaches. Mice were pre-immunized by IM injection of AAV2/GFP then challenged with AAV1, AAV3, AAV4, and AAV5 (20). Data showed that AAV2 antibodies only neutralized AAV3. Another model used an intravenous (IV) infusion in mice of AAV2 immunoglobulin from a pool of individual human donors prior to liver delivery of AAV2, AAV6 or AAV8 expressing hFIX. AAV2 administration was completely blocked while AAV6 and AAV8 were partially blocked (18). Given that the immunoglobulin pool was not assessed for AAV6 or AAV8 antibodies the results seen may not have been due to cross-reactivity.

#### **OVERCOMING HUMORAL RESPONSES TO AAV VECTORS**

A comparative approach to test the ideal route of administration to overcome pre-existing NAbs in mice involved an initial IV infusion of AAV2 immunoglobulin from a collection of AAV2 seropostive serum prior to administrating AAV2 via different routes. Delivery of AAV2 to liver by portal vein or direct injection into the liver parenchyma showed successful transduction in the face of preexisting AAV2 antibodies prophylaxis, while IV injection did not (21). It should be noted that even direct administration of vector to the liver resulted in lower expression levels in the presence of NAb, indicating that even this approach is not able to completely bypass Nab inhibition.

Manipulating the capsid is another approach to overcome NAbs. Generation of alternative capsid libraries by rational mutations in antigenic regions (22), error prone PCR (23), and DNA shuffling (24) have demonstrated the potential. PEGylation is a chemical modification which involves pre-coating the AAV capsid with polyethylene glycol (PEG) prior to administration and has shown promise at limiting but not preventing neutralization (25, 26). Another promising strategy involves artificially encapsulating the AAV vector in biomaterial prior to administration which has the potential to shield the AAV capsid (27). This may be useful provided the biomaterial cloak can be degraded after delivery. While these approaches are encouraging they may limit transduction efficiency.

Temporary immunosuppresion (IS) is emerging as a preferred approach for overcoming humoral responses and has been tested in pre-clinical studies. The inhibition of helper T-cells using CD4 antibodies or cyclosporine A prevents NAb formation and facilitates vector readministration (28). Additionally, transient B-cell depletion using Rituximab (CD20 antibody) can reduce preexisting antibodies (29). Plasmapheresis can eliminate pre-existing humoral responses but requires multiple cycles of blood exchange to reduce NAbs to negligible levels (30). Similarly, flushing the macaque liver with saline prior to delivery of vector limited NAb inhibition (31). In an ongoing clinical trial, empty capsids were added to the vector with the rational that they will bind away Nabs and thus increase transgene expression (32). However this approach involves increasing vector load, which may not be favorable for trying to prevent the development of CTL responses.

#### **CHARACTERIZING INHIBITORY CTL RESPONSES TO AAV IN PRE-CLINICAL STUDIES**

The first CTL response with parenchymal damage, loss of transgene expression, and expansion of capsid-specific CTLs were observed during a liver directed clinical trial for HB. This was unexpected since it was not observed in pre-clinical studies including non-human primates. Early attempts to replicate this immune response in animals were unsuccessful (33, 34). Even dog models have shown consistent transgene expression without an immune response (5). Therefore, efforts were made to determine variations in immunity between humans and pre-clinical animals. The absence of CTL responses in non-human primates, which are natural hosts of AAV8, ruled out the expansion of memory T-cells as the contributing factor (35, 36). Varki and colleagues reported the loss of siglec receptor expression in T lymphocytes during human evolution (37). Siglec receptors have inhibitory function on the immune system. Exploring the loss of these inhibitory receptors as a possible reason revealed enhanced T-cell function in a mouse KO model which mimics human T-cells, suggesting that human T-cells respond with more activation and proliferation (38). However, the response to AAV also could not be induced in these KO mouse.

In trying to develop CTL responses in pre-clinical animal models, one approach delivered a rAAV expressing IL-12 to the liver (39). Despite the induction of liver inflammation and generation of capsid-specific CTLs, transduced hepatocytes were not eliminated. Another model used an immunogenic OVA/SIINFEKL mouse system. The OT-1 mouse line has an increased frequency of OVA SIINFEKL epitope-specific CTLs. The model requires immunization of OT-1 mice with Ad-AAV. Splenocytes were harvested, labeled with carboxyfluorescein succinimidyl ester (cfse), and adoptively transferred into mice injected with an AAV vector with the SIINFEKL epitope incorporated into the capsid (40). Prolonged proliferation of OT-1 transferred cells (at least 10 weeks after transfer) was observed compared to transfer of wt splenocytes from Ad-AAV immunized mice, however no parenchymal cytotoxicity was reported. The Samulski lab also used the SIINFEKL mouse system. They discovered reduced transgene expression after adoptive transfer of OT-1 splenocytes however only in AAV-treated mice pre-immunized with SIINFEKL-pulsed DCs. Unfortunately, this approach is not ideal given the high immunogenicity, which might not fully reflect actual immunity (41).

#### **IN VITRO ASSAYS FOR CHARACTERIZING CTLs**

*In vitro* assays have had more success at characterizing AAV capsid CTL development and involve monitoring CTL activation and cytotoxicity in response to peptide presentation of AAV capsids in target cells. It is possible to detect AAV capsid-specific CTLs by staining with a multimeric antibody. However staining with these multimers only detects single T-cell clones and is HLA restrictive. As a result, T-cells only from individuals who express a specific HLA type can be visualized. An engineered cell line, which expresses luciferase upon engagement of a specific T-cell receptor was used to monitor T-cells activation (42). Again, this assay is restricted to one HLA type (HLAB0702) and AAV2 recognition. A more flexible assay measures cytotoxicity toward AAV-transduced hepatocytes. In this assay, different serotypes can be tested as long as they transduce the target cells (43, 44). Briefly, CTLs are generated by several rounds of stimulation and proliferation of target cell HLA matched peripheral blood mononuclear cells (PBMCs) or spenocytes with the AAV serotype of interest or dominant epitope and then co-cultured with target cells that are transduced with that AAV and cytotoxicity is measured. In general, a major limitation of *in vitro* assays is the fact that transduction efficiency of alternate AAV serotypes differs significantly and hinders comparative studies between alternate capsid variants and serotypes. Still these assays are helpful in monitoring CTLs responses in mice and in PBMCs obtained during clinical trials.

#### **VECTOR CAPSID VARIATIONS FOR AVOIDING CTL RESPONSES**

There is a growing field of vector alterations to increase efficiency and avoid immunity. The rational for creating capsid mutants differs among groups, however most target tyrosine and serine residues on the AAV capsid to enhance transduction efficiency. The Srivastava lab (45) has reported a triple tyrosine capsid mutant with increased transduction efficiency *in vitro* and *in vivo*. Other groups also report increased transduction efficiency by altering tyrosine residues on the AAV capsid (46). Initial data suggests that increased transgene expression is likely due to reduced proteasomal degradation which will shuttle more vector to the nucleus, but the intracellular trafficking of vector has not been fully characterized.

From an immunological perspective, avoiding proteasomal degradation may also lead to reduced CTL recognition. Tyrosine mutants were tested in an immunological mouse model and the group reported increased transgene expression and showed reduced T-cell immunogenicity and toxicity within the liver (44). To mimic the cellular immune reaction in humans, the group adoptively transferred *in vitro* expanded CTLs from AAV immunized mice. In this setting, they were able to demonstrate cytotoxicity in the liver by reduction of transgene levels and increased liver enzymes, both of which were reversed when using the capsid mutant.

#### **PHARMACOLOGICAL INTERVENTION**

General IS (taken from treatment of autoimmune disorders or transplantations) (9) and more specific interventions for AAV have been proposed and tested in pre-clinical models, such as proteasomal inhibitors (PI) (e.g., bortezomib, MG 132, carfilzomib). Many groups focus on increasing AAV transduction levels in various tissues using PI (47–50), and other compounds like arsenic trioxide (51), rather than their influence on the immune response. At the same time, another group observed reduced CTL responses as well (42). The mechanism is not completely understood yet it is hypothesized that AAV proteasomal capsid degradation is disrupted which leads to diminished capsid–peptide presentation by MHC class I molecules. This intervention has only been tested in pre-clinical systems for mechanistic value, given that the later fate of undigested capsids is unknown and could present the danger of prolonged or delayed capsid–peptide presentation.

#### **DEVELOPING TOLERANCE**

Multiple groups have demonstrated induced tolerance to the transgene for hepatic delivery of AAV vectors with the involvement of regulatory T-cells (Tregs) (52–55). Attempts to induce tolerance to the AAV capsid through the enrichment of Tregs have been published and show promising results. Initially, De Groot and colleagues were able to show reduced immune responses to common immunogens *in vitro* and *in vivo* using MHC class II epitopes (Tregitopes) that favor Treg development (56). These observations were extended to AAV capsid tolerance in a subsequent study (57). They show AAV capsid CTL suppression using these Tregitopes.

#### **CONCLUSION**

Due to the combined efforts of multiple groups working on the various aspects of immune responses to gene delivery with a rAAV vector, many parameters have been deciphered. For humoral immune response models, various models have been established to characterize pre-existing humoral immunity and cross-reactivity and assess the impact of potential interventions*in vitro* and *in vivo*. Using pre-existing KO mice, the influence of the innate immune system is also being rapidly investigated. In contrast, finding models to mimic and investigate the cellular immune response toward AAV capsids has caused more difficulty, only robust *in vitro* models have been well established but recently some features of the response could be recapitulated in animal models. It remains to be seen if these approaches will be able to provide a sufficient platform for further deciphering of cellular immune response and testing interventions. While characterizing immune responses in animal models have been informative, it is evident that greater emphasis should be placed on developing CTL models in immunocompetent animals that are more reflective of the clinical scenario. Until reliable models are established, investigators can use the valuable published data gathered in human clinical trials about cellular immune responses and their management (8, 9).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### *Received: 24 October 2013; accepted: 17 January 2014; published online: 07 February 2014.*

*Citation: Basner-Tschakarjan E, Bijjiga E and Martino AT (2014) Pre-clinical assessment of immune responses to adeno-associated virus (AAV) vectors. Front. Immunol. 5:28. doi: 10.3389/fimmu.2014.00028*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Basner-Tschakarjan, Bijjiga and Martino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Humoral immune response to AAV

#### **Roberto Calcedo and James M.Wilson\***

Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

#### **Edited by:**

Federico Mingozzi, UPMC, France

#### **Reviewed by:**

Thomas Weber, Icahn School of Medicine at Mount Sinai, USA Philippe Veron, Genethon, France

#### **\*Correspondence:**

James M. Wilson, Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, TRL, Suite 2000, 125 S. 31st Street, Philadelphia, PA 19104, USA e-mail<EMAIL_ADDRESS>upenn.edu

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.

**Keywords: adeno-associated virus, AAV, neutralizing antibody, immune response, gene therapy**

#### **INTRODUCTION**

Adeno-associated virus (AAVs) 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 is a small, non-enveloped single-stranded DNA virus that has been detected in many different tissues of several animal species (1, 2) but has not been associated with any disease (3, 4). In the past decade the discovery and development of new AAV types with dramatically improved *in vivo* performance and with unique seroreactivity and tissue tropisms (5–9) has situated AAV in the forefront of vector development for gene therapy trials. One of the most important aspects of the development of AAV as a clinical product is the impact of the host humoral immune response against its capsid. Several studies have shown that the induction of antibodies by natural exposure to AAV early in life can compromise the subsequent use of AAV as a gene therapy vector (10–14). Moreover administration of an AAV vector induces a potent and long term humoral response to AAV that may compromise the use of the same vector if a second administration is required (15, 16). Humoral immune responses to AAV can be of two types: neutralizing or binding (non-neutralizing). Neutralizing antibodies (NAbs) bind to AAV and through several mechanisms (17) inhibit AAV transduction of target cells. Non-NAbs bind to AAV and "flag" the virus without blocking AAV transduction. AAV NAbs have been the focus of many studies because of their significant deleterious effect on the efficacy of AAV-mediated gene therapy. Recent studies have shown that AAV binding antibodies may also have an impact on AAV vector distribution and safety (18).

In this review we will provide an overview of humoral responses to natural infection with AAV and to therapeutic AAV vectors in small and large animal models including humans. We will also discuss the best method to detect these antibody responses and summarize strategies that have been proposed to avoid or overcome NAbs to allow for AAV gene therapy in a wide spectrum of subjects or to patients that already received AAV-mediated gene therapy but need to be re-treated.

#### **DETECTION OF ANTI-AAV ANTIBODIES**

Several methods have been developed to detect antibodies to different AAV serotypes. Some of these methods detect total binding antibodies to AAV capsid and other methods detect antibodies that neutralize *in vitro* or *in vivo* transduction of AAV vectors. The first reports in the early 1970s evaluated total antibodies responses to AAV vector as measured by ELISA and Western blot (19–24). These studies focused on AAV1 and AAV2, as these were the AAV serotypes available at that time. The development in the last decade of new AAV types as delivery vectors for gene therapy required more sophisticated assays to evaluate the level of not only binding but NAbs specific to each AAV serotype.

The *in vitro* transduction inhibition assay became the standard assay to evaluate these NAbs to AAV. The assay is usually carried out in a 48- or a 96-well plate format allowing a high throughput sample analysis. Several cell lines have been used as targets for AAV vector transduction: HeLa, 2V6.11, 293, and Huh7 (25– 29). Typically an AAV vector expressing a reporter gene is mixed with serial dilutions of the test sample and the vector-serum mixture is incubated with the cell line of choice that is subsequently analyzed for reporter gene expression. The starting dilution of the test sample, which defines the sensitivity of the assay, varies between studies ranging from 1/2 to 1/20 (29–33). In some cases pre-infection of target cells with wild type adenovirus is included to increase AAV transduction. At the same time the AAV vector expressing the reporter gene is mixed with serial dilutions of naïve serum. The purpose of incubating the vector with naïve serum is to evaluate the enhancement in transduction observed at high concentration of serum ≤1/5 (18). The NAb50 titer is reported as the highest serum dilution that inhibits transduction by 50%. *In vitro* transduction efficiency is AAV serotype dependent. The high level of *in vitro* transduction observed with AAV2 combined with the use of very sensitive luminescence reporter genes allows the use of an AAV particle/cell ratio as low as 100 (18). On the other hand, the low levels of *in vitro* transduction observed in some of the new AAV serotypes, like AAV8, demands the use of a higher AAV particle/ratio which for AAV8 is between 1 × 103 and 1 × 104 (10, 18, 27, 30). The use of the minimum amount of AAV particles per cell for each AAV serotype allows the determination of the AAV NAb titer using the most sensitive assay but compromises the comparison of NAb titers between all AAV serotypes. Moreover, the high number of AAV particles/cell in addition to the full/empty AAV particle ratio of the AAV vector preparation used compromises sensitivity and the reproducibility of the results obtained between different laboratories. It should be noted that this assay does not consider the potential antibodydependent cellular phagocytosis (20) as a blocking mechanism of vector neutralization or the possible change in tissue targeting of AAV (18).

To overcome the above mentioned problems, an *in vivo* NAb assay has been developed by various investigators (10, 18, 34). In this assay, mice are infused with the serum sample immediately prior to intravenous infusion of the AAV vector expressing a secreted reporter gene. Since the level of transduction by the new AAV serotypes is superior to AAV2 in most *in vivo* applications a dose as low as 109 GC/animal can be used. Several secreted transgenes have been used to evaluate transduction including FIX and α-galactosidase (10, 18). A reduction in transgene expression of 50% or more when compared to control mice injected only with AAV vector is reported as positive for AAV NAbs. This assay is more sensitive than the *in vitro* assay as up to 29% of samples that were negative by the *in vitro* NAb assay scored positive for AAV NAbs (10, 18). The problem with this assay is that some of the monkeys that tested positive for AAV NAbs did not show a reduction in AAV-mediated gene transfer, suggesting the *in vivo* NAb assay is too sensitive (18).

The conclusions of these studies suggested that the *in vitro* NAb assay provides a better correlation with *in vivo* transduction in macaques than the *in vivo* NAb assay. Therefore the *in vitro* NAb assay has become the standard assay to evaluate clinical samples for the presence of AAV NAbs prior vector administration.

#### **ANTI-AAV ANTIBODIES IN SEVERAL NON-PRIMATE SPECIES**

Adeno-associated virus has been isolated from several tissues of non-primate animal species including rat, mice, sheep, bird, snake, cows, goat, and pig (2, 7, 24, 35–43), suggesting a natural exposure to AAV. Pre-existing Abs to AAV in these non-primate species were not thought to be a problem because endogenous parvoviruses were believed to be structurally distinctfrom primateAAVs.Analysis of serum from small and large non-primate species used as pre-clinical animal models has shown high rates of detectable levels of NAbs to several AAV serotypes found in both monkeys and human. Interestingly the prevalence of AAV NAbs is both AAV serotype and species specific (44). In horses, AAV5 is the most seroprevalent serotype with 100% of the samples testing positive for NAbs. In dogs, AAV6 is the most seroprevalent serotype with 100% of the samples positive for NAbs (28, 45). Interestingly, high levels of AAV6 NAbs are found in newborn puppies suggesting passive immunization from colostrum and breast milk

(45). 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. A recent study, also in dogs, showed the high binding capacity of serum protein human galectin 3 binding protein to AAV6 inducing the formation of aggregates and hampering vector transduction (46). In pigs, AAV5 is the most seroprevalent AAV serotype with 100% of the samples testing positive for NAbs; seroprevalence of NAbs to other serotypes like AAV1, AAV2, and AAV8 was close to 50%. AAV6 was the least seroprevalent serotype in pigs. *In vivo* testing of serum from these species confirmed the neutralizing activity of these antibodies (28, 44). These studies highlighted the importance of AAV NAb screening of all animal models used to evaluate *in vivo* AAV performance for pre-clinical gene therapy.

#### **ANTI-AAV ANTIBODIES IN PRIMATES AND HUMANS**

Prevalence of antibodies to AAV in humans was first reported in early 60s and 70s and mainly focused on AAV1 and AAV2, the only serotypes available at that time. Frequencies of antibodies ranged from 30 to 80% among human populations (19, 21–23). Recently, more than 100 natural AAV variants have been isolated from human and non-human primates tissue specimens (2, 6, 41). In pre-clinical models AAV7, AAV8, AAV9, and AAVrh.10 have emerged as promising candidates for gene therapy quickly becoming the most commonly used AAV serotypes in pre-clinical research. Several studies addressed the prevalence of NAbs to these new serotypes and compared them to previously described serotypes (25, 29, 30, 32, 47). In all human populations studied, which included samples from 10 countries and four continents (America, Europe, Africa, and Australia), prevalence of NAbs to AAV2 ranged from 60 to 30% and was significantly higher than the prevalence of NAbs to AAV7,AAV8, and AAV9 serotypes which ranged from 30 to 15%. Although the seroprevalence of NAbs to AAV1 was lower than that for AAV2, it was still higher than AAV7, AAV8, and AAV9 in most regions. It is worth noting that significantly higher frequencies of NAbs to all AAV serotypes were observed in Africa (25). Interestingly, the closely related AAV4 and AAVrh32.33 serotypes showed the lowest seroprevalence with less than 2% of the population testing positive worldwide (25, 35). Although these properties were encouraging for the development of AAVrh32.33 as vector for gene therapy, recent studies have shown this serotype induces a remarkable strong T cell mediated immune responses to the transgene product, similar to that induced by adenovirus, considerably limiting gene expression duration (48). Interestingly, when the seroprevalence to AAV serotypes was analyzed in non-human primates, including rhesus macaques, cynomolgus macaques, Japanese macaques, pig tail macaques, squirrel monkeys, chimpanzees, and baboons, AAV7, AAV8, AAV9, and AAVrh10 were the serotypes with the highest seroprevalence, with NAb frequencies up to 100% (6, 49, 50). Contrary to the situation in humans, AAV2 became the AAV serotype with the lowest seroprevalence (51). This finding is supported by

the fact that AAV7, AAV8, and AAVrh10 were originally isolated from rhesus macaque tissues (2, 7). The high seroprevalence of these AAV serotypes in non-human primates presents an important challenge in the evaluation of vector performance in this animal model and an exhaustive screening of monkey colonies for pre-existing NAbs is required.

Binding AAV antibodies may also play an important role in the clinical application of AAV vectors. AAV particles opsonized by non-NAbs may be taken up by cells of the immune system such as dendritic cells and macrophages through Fc receptors which may lead to the development of inflammatory responses. Frequencies of binding antibodies in humans were close to 70% for both AAV1 and AAV2, 45% to both AAV6 and AAV9, and 38% for AAV8 (30). Although the frequencies to binding antibodies were higher than frequencies of NAbs, the relative frequency between AAV serotypes remained the same with AAV1 and AAV2 being the most prevalent AAV serotypes.

Several studies have tried to associate binding antibodies to NAbs with the goal of using binding antibodies as an indirect measure of NAbs (10, 18). Although these studies found a correlation between both types of antibodies, almost 20% of the samples with high binding antibodies had very low or no detectable NAbs. The significance of the Fc interactions to host cells/proteins has been demonstrated in a large animal study in which AAV8 was administered systemically in a non-human primate model with pre-existing AAV8 antibodies. The study showed that AAV vectors were redirected from the liver to the spleen where they were stably sequestered by follicular dendritic cells (18). Although pre-existing humoral immune responses to the AAV capsid do not always correlate with the presence of AAV capsid deposition in the spleen this finding raised concerns of the safety profile of systemic AAV administration.

#### **CROSS-REACTIVITY OF THE ANTIBODY RESPONSE**

An important feature of the humoral response against AAV is the breadth of the response. If a subject is positive for antibodies to a specific AAV serotype, what is the likelihood that this subject will also be positive for other AAV serotypes? Several studies have analyzed the specificity of this response and found a strong link in seropositivity toward distinct AAV serotypes. The majority of the subjects with NAbs and/or binding antibodies to AAV7,AAV8, and AAV9 also had antibodies to AAV2. Conversely, only a few subjects with NAbs and/or binding antibodies to AAV2 had also antibodies to AAV7, AAV8, and AAV9 (25, 30).

Most subjects enrolled in the recent phase 2 clinical trial of AAV1 vector expressing α1-antitrypsin developed a NAb response specific to AAV1 with minimal or no cross-reactivity to AAV2, AAV7, and AAV8 serotypes (15, 52). Only those subjects with low pre-existing NAbs to AAV1, AAV2, AAV7, and AAV8 developed a highly cross-reactive NAb response to AAV2, AAV7, and AAV8 when injected with AAV1. One hypothesis to explain the difference in the breath of the AAV NAb response between naturally exposed and gene therapy-treated subjects is that subjects with co-occurrence of NAbs against multiple AAV serotypes may be the result of multiple infections with various AAV types. Moreover the propensity of AAVs to evolve through various mechanisms of molecular evolution (6) would lead to the generation of NAbs

directed against a wide spectrum of homologous antigens. The fact that AAV2 is the serotype with the highest prevalence in the human population and also with the highest NAb titer indicates that the initial and most frequent exposure to AAV in humans occurs with an AAV2 or an AAV2 like serotype (31).

This data would also indicate that subjects naive for AAV NAbs would be the preferred candidates for gene therapy if a second administration of AAV vector is required. These subjects may develop a NAb response specific to the first AAV serotype injected, and a second administration with a different AAV serotype should not pose a problem because of the narrow breath of the AAV NAb response generated. Subjects with pre-existing NAbs to multiple serotypes with a AAV NAb titer low enough (≤1/10) not to interfere with the first vector administration would develop a strong and broadly cross-reactive AAV NAb response that may block a second vector administration with another AAV serotype.

#### **AGE, GENDER, HEALTH STATUS, AND GEOGRAPHICAL REGION DEPENDENT PREVALENCE OF NAbs**

When and how humans are exposed to AAV for the first time is still not clear, although several studies suggest that this may happen early in life as serum-circulating binding and NAbs have been reported in children (44, 53). This is especially important for the treatment of many genetic diseases that manifest early in infancy and therefore early gene therapy treatment with AAV is beneficial. NAbs to AAV2 were detected in almost 60% of the infants and to AAV8 in 36% of the infants right after birth. Prevalence of NAbs to both AAV serotypes declined during the first year of life due to the drop in maternal antibody levels. A continuous increase in the prevalence of AAV NAbs after 1 year of age with a peak at 3 years of age suggests this age window is the time of the first exposure to natural AAV infections, and closely models that of adenovirus infection (22). Therefore the best age for an early gene therapy intervention with an AAV vector may be right prior to 1 year of age. The higher prevalence of AAV2 NAbs over AAV8 in early childhood also confirms the hypothesis that AAV2 or an AAV2 like serotype is the first AAV to which humans are exposed.

Prevalence of AAV NAbs can vary depending on the geographical origin of the population studied.While the prevalence of AAV1 NAbs in Africa and China is close to 50–70%, in other countries like Belgium, Greece, Italy, and USA it is only 20–30% (25, 54). Overall the prevalence of AAV NAbs seems to be higher in developing countries. It remains unclear whether living conditions, population density, hygienic conditions, different level of health care, MHC background, or method of detecting AAV NAbs are involved in this phenomenon. Interestingly, gender is another factor that influences the prevalence of AAV NAbs. Women have a significantly higher prevalence of AAV1 NAbs than men (54). The health status of the target population may also impact the prevalence of AAV NAbs, especially in those subjects with a compromised immune system. These subjects had a lower prevalence of AAV NAbs when compared to the healthy population (32, 55).

#### **STRATEGIES TO OVERCOME AAV NAbs IN AAV-MEDIATED GENE THERAPY**

The presence of AAV NAbs in both animals and human subjects has necessitated the development of strategies to generate new AAV variants with limited or reduced recognition by NAbs. One approach has focused on the identification of the immunogenic domains on the AAV capsid and their subsequent modification to avoid recognition by NAbs. Such targeted engineering of AAV capsid requires knowledge of the antibody recognition site, the epitope. At present, only epitopes to AAV2 and AAV8 serotypes have been identified (56, 57). In this regard, it should be noted that strategies to determine epitopes that are conformational are likely to be more challenging. In spite of these difficulties, it has been demonstrated that amino acid substitutions at sites 459, 493, 551, and 587 of AAV2 capsid confer escape from neutralizing human antisera. While modification of these epitopes provided a reduction in neutralization, it did not completely avoid it. Naturally occurring variants with different amino acid composition at these sites outperformed these mutants in terms of eluding NAbs (8).

An alternate approach is the development of an AAV capsid library generated by error-prone PCR that is subsequently screened in the presence of NAbs to select AAV variants resistant to neutralization. This technique, called directed evolution of AAV vectors, has been employed by Maheshri et al. to identify an AAV2 variant that carried the E12A, K258N, T567S, N587I, and T716A mutations which was 96-fold more resistant to neutralization by anti-AAV2 polyclonal serum than wild type AAV2 (9). A similar approach, but using human serum positive for AAV2 NAbs, identified an AAV2 variant containing R459K and N551D point mutations which was 5.5-fold more resistant to neutralization than wild type AAV2 (58).

The creation of these new AAV variants and the discovery new AAV serotypes (2, 7) with impressive transduction capabilities and distinct serological properties have provided scientists with the tools necessary to avoid undesirable NAbs to one AAV serotype by using a different AAV vector type. This approach has been shown to be effective in both small and large animal models (16, 59). Although this approach is viable for subjects with an AAV NAb response restricted to one serotype or with a cross-NAb titer equal or less than 1/10 (18) it may not be in those subjects with high titers of cross-reactive NAbs. To overcome this problem several strategies have been proposed:

vector in the target organ and minimizing the exposure of the vector to circulating NAbs has been shown to be efficient with AAV8 in non-human primate studies targeting the liver via the portal vein (26). In this study investigators flushed the liver with saline, to remove blood and NAbs with it, before injection of the vector into the portal vein. Their results showed no significant impact on gene expression when animals had an AAV NAb titer of up to 1/28 and only partial reduction of gene transfer when the NAb titer was 1/56.

Another approach to minimize the contact of AAV vector with NAb includes targeting a tissue or organ via direct infiltration rather than via the circulation. This organ/tissue should have a proven record of stable and long term AAV-mediated gene expression. Muscle has been one of the candidate sites to test this approach. Investigators using non-human primates and an AAV8 vector have shown minimal impact on systemic gene expression in the presence of AAV8 NAbs as high as 1/320 when monkeys were injected directly into muscle (61). A similar approach has been used in clinical trials for A1AT deficiency and hemophilia B using AAV1 and AAV2 vectors respectively, demonstrating that high titers of AAV NAb did not prevent gene transfer (15, 52, 62). The retina has also been used as a target organ for AAV-mediated gene therapy for localized systemic expression of therapeutic proteins such us erythropoietin (63). As into the muscle, the subretinal space into which the vector is injected has reduced contact with blood; consequently pre-existing AAV NAbs in blood had a minimal impact of AAV-mediated gene transfer (64, 65). Intrathecal administration (i.e., direct injection into the cerebral spinal fluid) is another route of administration that minimizes contact of the AAV vector with blood. In recent mouse studies, AAV2 and AAV5 were injected intrathecally to transduce the central nervous system (66). These studies showed that the potentially debilitating effect of AAV NAbs could be partially overcome by direct administration of AAV to the target organ. The caveat associated with these alternative routes of administration is the immune response that is induced in these organs/tissues. For example, the muscle is a very immunogenic tissue and intramuscular administration, unlike intravenous administration, induces a strong humoral and cellular immune response to the AAV capsid and in some instances to the transgene product (15). Humoral and cellular immune responses to the AAV capsid and transgene product using these alternative routes of administration need to be carefully assessed to ensure that the lack of AAV interference with pre-existing NAbs is not eclipsed by the induction of a stronger immune response to the therapeutic product.

(c) Immunosuppression has been proposed by several groups as a method to lowering NAbs by reducing the number of cells producing antibodies. In these studies rituximab, a B-cell depleting antibody that targets the CD20 antigen and is used clinically, was able to reduce AAV2 and AAV5 NAbs in ∼30% of subjects to levels that for some subjects were under limit of detection (32). These data are consistent with results obtained in non-human primates using rituximab and cyclosporine, although in this study rhesus

macaques were injected systemically with an AAV vector and then NAb response were monitored (67). The caveat with pharmacological immunomodulation of the immune response is that the tolerogenic properties of AAV can be altered after this treatment and undesirable immune response to the transgene may be induced, as was reported for monkeys injected with AAV2 and carrying FIX as transgene (68). An immediate antibody response to the FIX was observed when the immunosuppression regimen was stopped after 10 weeks of vector administration. An antibody response to FIX was never observed in monkeys that did not receive immunosuppression.

#### **REFERENCES**

In conclusion, a combination of alternative AAV types, route of vector administration with minimum contact with blood and techniques directed to lower AAV NAb by physical methods or pharmacological modulation of the humoral immune response may ultimately overcome the impact of pre-existing AAV NAbs in subjects who would otherwise not be eligible for AAV-mediated gene transfer therapy.

#### **ACKNOWLEDGMENTS**

The authors would like to thank Maria P. Limberis and Suryanarayan Somanathan for their insightful comments and careful review of the manuscript.

*Gene Ther* (2011) **22**(10):1239–47. doi:10.1089/hum.2011.053

define two new AAV species distantly related to AAV-5. *Virology* (2006) **353**(1):68–82. doi:10.1016/j. virol.2006.05.023

of adeno-associated virus. *J Gene Med* (2006) **8**(2):155–62. doi:10. 1002/jgm.849

primate model of AAV gene transfer for hemophilia B. *Mol Ther* (2012) **20**(7):1410–6. doi:10.1038/ mt.2012.84

68. Mingozzi F, Hasbrouck NC, Basner-Tschakarjan E, Edmonson SA, Hui DJ, Sabatino DE, et al. Modulation of tolerance to the transgene product in a nonhuman primate model of AAVmediated gene transfer to liver. *Blood* (2007) **110**(7):2334–41. doi: 10.1182/blood-2007-03-080093

**Conflict of Interest Statement:** James M. Wilson is a consultant to ReGenX Holdings and is a founder of, holds equity in, and receives a grant from affiliates of ReGenX Holdings; in addition, relevant to this work, he is an inventor on patents licensed to various biopharmaceutical companies, including affiliates of ReGenX Holdings. Roberto Calcedo declares no competing interests. James M. Wilson holds a patent on adeno-associated virus (AAV) clades (U.S. Patent 7,906,111B2) with pending continuation (U.S. Patent 13/023,918).

*Received: 08 July 2013; accepted: 05 October 2013; published online: 18 October 2013.*

*Citation: Calcedo R and Wilson JM (2013) Humoral immune response to AAV. Front. Immunol. 4:341. doi: 10.3389/fimmu.2013.00341*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Calcedo and Wilson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Mapping the AAV capsid host antibody response toward the development of second generation gene delivery vectors

#### **Yu-Shan Tseng and Mavis Agbandje-McKenna\***

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA

#### **Edited by:**

Federico Mingozzi, UPMC Paris 6, France; Genethon, France

#### **Reviewed by:**

David Schaffer, University of California Berkeley, USA Hildegard Büning, University of Cologne, Germany

#### **\*Correspondence:**

Mavis Agbandje-McKenna, Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA e-mail<EMAIL_ADDRESS>
The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape.Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes.The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

 They are small (~26 nm) non-enveloped viruses belonging to the *Parvoviridae*, are assembled with *T* = 1 icosahedral capsid symmetry, and package a 4.7 kb single-stranded (ss) DNA genome (1). There are over 100 AAV genomic isolates and 13 human and non-human serotypes described. These viruses have different transduction efficiencies in different tissues dictated by the capsid sequence (2). To date, no diseases have been associated with wild-type AAV infection. Further, AAVs can transduce both dividing and non-dividing cells and sustain long-term gene expression in non-dividing cells (3). All these properties make them desirable vectors for therapeutic gene delivery.

Recombinant AAV (rAAV) vectors, used in clinical trials, contain a desired transgene cassette flanked by two inverted terminal repeats (ITRs) instead of the wild-type viral genome flanked by these elements (4). In recent years, the AAV gene delivery system has been successfully utilized in several animal and human clinical trials. In an ongoing hemophilia B trial, therapeutic levels of Factor IX protein has been maintained in patients for over 2 years with only one infusion of an rAAV8 vector packaging this gene (5). In addition, rAAV2 vectors, encoding the retinal pigment epithelium-specific 65 kDa protein, improved vision in Leber's congenital amaurosis (LCA) patients, without any significant side effects (6–9). rAAV vectors have also been developed for the transduction of a variety of other cells in addition to liver and the eye, including, as examples: brain cells for the treatment of Parkinson's disease (10) and Canavan disease (11); skeletal muscle for the treatment of emphysema, lipoprotein lipase deficiency, and muscular dystrophy (12–14); and heart muscle for the treatment of heart failure (15). Significantly, in 2012, an AAV1 vector, encoding the lipoprotein lipase, was approved as a gene therapy treatment in Europe (16), heralding a new era for this vector system.

However, despite the above successes, several obstacles must still be overcome for full realization of the AAV vector system in patient care and treatment. One of the most important of these is pre-existing immunity. Serologic studies have shown that the majority of the human population has been exposed to wild-type AAVs (17–19). For example, the prevalence of anti-AAV antibodies in the human population has been reported to be ~40–70%, with the most reactivity against AAV2, the most studied and best characterized of the AAV serotypes. Although rAAV vectors used for gene delivery do not carry viral genes and are unable to drive viral protein synthesis, they are assembled from wild-type viral capsid shells; thus, the host immune response to the vector can be influenced by prior exposure to wild-type AAV. A pre-existing

antibody response against AAV can initiate an immune memory response, which could impede gene delivery. For example, neutralization effects from pre-existing antibodies have been reported to decrease transduction efficiency, even at low antibody titers (20– 22). For this reason, in the recent gene delivery trial for hemophilia B, individuals with evidence of pre-existing AAV antibody immunity were excluded from participation (5). Thus, to develop the AAV vector system into a more practical and efficacious gene transfer system, it is important to understand how antibodies interact with the AAV capsid, especially to map dominant epitopes, both neutralizing and non-neutralizing. With sufficient information on the AAV antigenic structure, combined with data on capsid determinants of tissue tropism and transduction, it would be feasible to design a neutralization-escaping vector, which can evade the host antibody immune response while retaining desired tissue tropism and transduction efficiency.

Here we will briefly review the AAV capsid structure and what is known about the effects of its interaction with antibodies, and then discuss example approaches utilized for mapping capsid antigenic epitopes. The three most common approaches include: directed evolution, an indirect method for obtaining the antigenic information through the selection of AAV variants under antibody pressure; epitope searching, which utilizes peptide scanning, peptide insertion, or site-directed mutagenesis; structural biology, namely cryo-electron microscopy and three-dimensional (3D) image reconstruction (cryo-reconstruction), which directly visualizes the antigenic sites on the capsid by 3D reconstruction of capsid-fragment antigen binding (Fab) complexes.

#### **AAV CAPSID STRUCTURE**

The ~4.7 kb genome of the AAVs contains three open reading frames (ORFs), *rep*, *cap*, and *aap*, flanked by two ITRs (~145 kb). This genome is packaged into *T* = 1 icosahedral capsids that are ~26 nm in diameter. The *cap* ORF encodes three overlapping structural capsid viral proteins (VPs): VP1, VP2, and VP3, in a ratio of 1:1:10, which assemble the capsid. A total of 60 VPs assemble the capsid by 2-, 3-, and 5-fold symmetry-related interactions (**Figure 1**). TheVP3, the major capsid component, is able to assemble the capsid as long as the assembly activating protein (AAP) encoded by the *aap* ORF, is present (23, 24). Three-dimensional structures have been determined for AAV1-AAV9, the clade and clonal isolate representatives of the over 100 genomic sequences known for the human and non-human primate AAVs (2), by either X-ray crystallography and/or cryo-reconstruction (25–32). In all these structures only the VP3 common sequence is ordered. While the AAVs have a sequence similarity that ranges from ~55 to 99%, they are structurally very similar (33). The VP topology consists of a conserved alpha helix (αA) and a eight stranded anti-parallel (βB-βI) β-barrel core with large inter-strand loops (**Figure 2A**) that form the exterior surface of the capsids. A comparison of the AAV2 and AAV4 structures, two of the most distantly related, identified nine common variable regions (VRs), designated VR-I to VR-IX (**Figures 2A,B**), located on the capsid surface at the top of the inter-strand loops (26, 34). The AAV capsid surface is characterized by depressions at the 2-fold axes (dimple), surrounding a cylindrical channel at the 5-fold axes (canyon), and protrusions surrounding the 3-fold axes (**Figure 1**). A wall or plateau is located

between the depression at the 2-fold axis and surrounding the 5-fold channel, the "2/5-fold wall" (**Figure 1**) (35). The VRs contribute to local topological differences between the AAV capsid surfaces. For example, VR-II forms the top of the 5-fold channel; VR-IV,V, and VIII form the top of the 3-fold protrusion and VR-VI and VR-VII form their base; and VR-I, III,VII, and IX contribute to the 2/5-fold wall (**Figure 2B**). The VRs also dictate functional differences, including receptor attachment, transduction efficiency, and antigenic reactivity between the AAVs (26, 28, 30, 36–39).

#### **AAV AND ANTIBODIES**

All viral vectors are susceptible to the immune response from the host (41). The most detrimental immune threat that AAV vectors encounter soon after administration is the B-cell mediated antibody response (42, 43). Antibodies use their complementarity determining region (CDR), located on the end of Fab region, to interact with antigens by specific surface complementarities (44). This binding site, or "epitope," is generally located on the capsid surface of viruses. Antibodies against viruses may neutralize infectivity prior to viral attachment to host cell receptors, or post attachment; interfering with internalization or fusion at the cell surface, or during endosomal trafficking (45–48). Other antibody neutralization mechanisms include antibody-mediated phagocytosis, complement binding and activation, opsonization, and antibody-dependent cellular cytotoxicity (ADCC) of infected cells (49–52). As already stated above, in humans the prevalence of AAV antibodies in healthy individuals is high and ranges from ~40–70% depending on serotype. Previous reports indicate that the highest prevalence of anti-AAV immunoglobulin G antibodies in humans was for AAV2 (~60–70%) and AAV1 (~35–70%), followed by AAV9 (~50%), AAV6 (~50%), AAV5 (~40%), and

AAV8 (~40%) [e.g., Ref. (17, 53)]. For the AAVs, neutralization mechanisms have been described for only three monoclonal antibodies (MAbs), A20 and C37-B against AAV2, which act at the post-entry steps and receptor attachment, respectively (54, 55), and ADK8 against AAV8, which acts at a post cell/pre-nuclear entry step (37).

Given the reported detrimental effects of anti-AAV antibodies on transgene expression, it is not surprising that the most successful applications of the AAV vector system have been in the eye and brain, immuno-privileged sites when vector is directly injected, or in patients with low or no anti-AAV antibodies titers prior to vector administration. Furthermore, since the AAV capsid structures are similar, different anti-AAV antibodies may cross-react, as has been observed, for example, between AAV2 and AAV3 that are highly similar at the sequence level (55), and recently for AAV1 and AAV5 which are disparate in at the sequence level (56). Boutin et al. (17) showed that patients with a positive AAV2 serum response were also seropositive for AAV1, AAV5, AAV6, AAV8, and AAV9 in 93, 52, 59, 57, and 58%, respectively, of the samples tested. Thus a new generation of AAV vectors is needed to circumvent the neutralization effects from pre-existing antibodies. The development of these new vectors will largely depend on available AAV antigenic and structure information. To obtain the antigenic information, three main approaches have been used. These are discussed below.

#### **DIRECTED EVOLUTION**

Without knowledge of AAV capsid biology, structure, or immunogenic sites, directed evolution serves as a strategy to generate neutralization-escaping AAV variants. It is a high-throughput molecular engineering procedure, which mimics natural evolution through iterations of genetic diversification under artificial selection pressure (57). For generating neutralization-escaping AAV variants, wild-type *cap* genes, from one or several AAV serotypes, are mutated to generate a large genetic plasmid library, which can generate numerous capsid variants through recombination during plasmid transfection. During viral infection with viruses arising from the recombination process, a selective pressure, in this case antibodies, is applied. Only the variants that can circumvent the antibody barrier presented to infect the desired cells will transduce and drive progeny virus synthesis. The successful variants are then recovered and amplified for the next round of selection. After several cycles of selection, additional mutagenesis can also be introduced before further selection to increase viral fitness (58). Comparison of the capsid sequences of the final resulting variants and the wild-type input viruses provides the antigenic information and the effect of the changes on tropism and transduction efficiency. Generally, there are two strategies to create the genetically diverse library, one is using error-prone mutagenesis to randomize the capsid DNA sequences; the other is to shuffle the DNA sequences of different AAV serotypes.

#### **ERROR-PRONE MUTAGENESIS**

By a "sloppy" polymerase chain reaction (PCR), random point mutations can be introduced into an ORF at a certain rate. Tuning the PCR conditions can also introduce a different number of mutations into the target gene sequence (59). Perabo et al. (60) used this approach to generate an AAV2 *cap* mutant library for directed evolution under human sera selection. Approximately, 70% of the neutralization-escaping variants obtained contained point mutations clustered on the external face of the capsid at the 3-fold protrusion (**Figure 1**). The two most frequently selected mutations were amino acids 459 and 551 (**Table 1**) located in AAV VR-IV and VR-VII, respectively (**Figure 2**). The variant with the best neutralization escape capability carried the double mutation R459K/N551D. The single (R459G or N551D) and the double (R459K/N551D) mutant variants had comparable genome packaging, infectivity, and particle titers to wild-type virus. However, the N50 value, the amount of serum required to decrease

#### **Table 1 | Antigenic sites identified by polyclonal antibodies.**

transduction by 50%, for R459G, N551D, and R459K/N551D were 4.1-, 3.3-, and 5.5-fold higher, respectively, than the corresponding N50 value obtained for the wild-type AAV2.

Maheshri et al. (61) improved this strategy by combining the error-prone PCR with a staggered extension process, which utilized a short time polymerase-catalyzed extension (64) to produce AAV2 variants. Mutant capsids were next selected for infectivity in HEK293 cells in the presence of a neutralizing rabbit anti-AAV2 serum. Nine mutants, which contained different combinations of mutations at amino acid positions 12, 42, 117, 152, 180, 258, 418, 493, 567, 587, 713, or 716, had neutralizing titers that were 3-fold higher than the wild-type virus (**Table 1**) (61). All the successful variants carried a T716A mutation. This residue is located on the capsid surface at the 2/5-fold wall next to VR-IX (**Figures 1** and **2B**). One variant, r2.15, had a 96-fold improvement in transduction compared to wild-type AAV2 and the ability to mediate moderate gene delivery at a low 1:2 serum dilution. This variant has two mutations not present in the others: T567S and N587I. T567S is in a minor epitope of the A20 MAb previously identified by pepscan and peptide competition (55). Residue 587 is in VR-VIII (**Figure 2**) and it is located proximal to residues involved in heparan sulfate proteoglycan (HSPG) receptor binding in AAV2 (65, 66).

Maersch and colleagues (67) also used error-prone PCR to establish an AAV2 *cap* library with the mutations focused only on amino acids located in the 3-fold region previously identified as being immunogenic: 449, 458, 459, and 551 (60), and 493 (61). The directed evolution was carried in HEK293 cells under the selection of neutralizing human serum. The resistance to neutralization of six resulting variants was compared to those of wild-type AAV1, AAV2, and the double mutant R459K/N551D generated by Perabo et al. (60). Two of the variants, with substitutions at 459, 493, and 551, outperformed the best variants from the previous selections by Maheshri et al. and Perabo et al. (60, 61). This observation indicated that fine tuning single amino acid types on the capsid surface can dramatically change the immunogenicity of an epitope. Residue 493 is located in VR-V, which as previously mentioned, together with VR-IV and VR-VIII form the top of the protrusions surrounding the 3-fold axis. Thus the data from errorprone mutagenesis provides indications that the 3-fold region is immuno-dominant in the AAVs.

#### **DNA SHUFFLING**

DNAse digestion followed by polymerase ligation can generate a chimeric capsid variant from different AAV serotypes. Grimm et al. (68) applied this method to randomly combine the cap sequences from AAV2, AAV5, AAV8, AAV9, caprine AAV, avain AAV, and bovine AAV prior to selection. They first selected capsid variants capable of human liver cell transduction and then selected in the presence of pooled human anti-sera (Intravenous immunoglobulin, IVIG). This strategy created a single chimera named AAV-DJ. The amino acid sequence for AAV-DJ was closely related to AAV2, AAV8, and AAV9 at 92, 88, and 85% identity, respectively. Interestingly, the IVIG selection generated this variant with higher homology to AAV8 compared to variants selected for human liver cell transduction alone, which were mostly similar to AAV2. This implies that the IVIG selection pressure eliminated variants with AAV2 epitope(s) from the resulting clones, consistent with the higher percentage of anti-AAV2 sero-prevalence in the human population compared to other AAV serotypes. AAV-DJ was capable of transducing mice passively infused with IVIG prior to infection at low (4 mg) IVIG dose to similar levels as parental AAV8 and AAV9 while AAV2 transduction was abolished in either high (20 mg) or low (4 mg) IVIG dose. The AAV-DJ was also inhibited at the high IVIG dose.

Another group, Koerber et al. (69), also generated chimeric AAV capsids through DNAse I digestion and polymerase ligation which displayed neutralization escape capability. The library was established from the *cap* ORF of AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9. The selection was carried once in HEK293 cells to optimize just for infectivity without any antibody pressure. However, surprisingly, some IVIG neutralization-escaping variants were generated. Four of the final seven chimeric capsids obtained showed improved IVIG neutralization resistance compared to the parental serotypes despite ~90% sequence similarity. Three of the variants that had higher resistance than the parental AAV2 had a ~80 aa stretch from AAV9 or a V709I mutation and the last C-terminal 19 aa from AAV6. The C-terminal stretch contained a previously described AAV2 epitope, 697–716 (AAV2 numbering, see below) (62). One of the chimeras, cB4, was similar to AAV1/6 at the sequence level, and had >400-, 8-, and 2-fold resistance to IVIG neutralization compared to AAV2, AAV1, and AAV6, respectively. While it was difficult to define which region

of this chimera dictated its strong immune evading ability, it contained a Y706H substitution. Both the Y706H and V709I changes would be located in the 2/5-fold wall.

Other examples of chimeras selected for infectivity without antibody selection which display increased antibody neutralization resistance have been reported. Li et al. (70) used iterative cycles of infection to select variants with hamster melanoma cell tropism started from a shuffled library constructed from AAV serotypes 1–9, except serotype 7. In this study, a specific chimeric AAV variant was isolated, containing residues 1–409 from AAV1, 410–450 from AAV8, 451–704 from AAV2, and 705–736 from AAV9. This chimera, chimeric-1829, did not show any cross-reactivity to antisera from mice immunized with AAV1, AAV8, and AAV9. The sera from mice immunized with AAV2 had some cross-reactivity to chimeric-1829, but the neutralizing antibody (Nab) titer was 25 fold lower than the titer for wild-type AAV2 (70). In another study, Yang et al. (71) used DNA shuffling and *in vivo* selection to generate a muscle-cell targeting AAV chimera from serotypes 1–9 with a different antigenic reactivity compared to the parental serotypes. The mutant, M41, assembled from AAV1, AAV6, AAV7, and AAV8 sequences, showed a higher resistance to IVIG neutralization compared to AAV2. At a 1:64 dilution of IVIG, AAV2 infectivity decreased to ~33%, whereas M41 infectivity still remained at 83% compared to controls without IVIG incubation (71). The wildtype AAV8 infectivity remained at ~94% under these conditions, suggesting that the sequence of M41 derived from AAV1, AAV6, and AAV7, not those from AAV8, resulted in the increased susceptibility to IVIG recognition. This observation is consistent with the fact that residues 410–450 contributed to M41 by AAV8 are mostly internal in the capsid and form core β-strand regions of the VP structure.

#### **EPITOPE SEARCHING**

Rather than viral evolution in the presence of antibodies to obtain information on the antigenic regions of the capsid, epitope searching focuses directly on the interaction(s) between antibodies and peptides generated from the capsid protein sequence to map possible epitopes. This approach utilizes three main strategies, peptide scanning, peptide insertion, and site-directed mutagenesis.

#### **PEPTIDE SCANNING**

Moskalenko et al. (62) scanned the entire AAV2 capsid amino acid sequence for potential epitopes using a total of 91 15-mer peptides that overlapped by five amino acids. The peptides were tested for their ability to inhibit capsid binding by AAV2 neutralizing human serum samples in an ELISA assay in which the peptides were applied to an AAV2 capsid coated ELISA plate. This study identified several overlapping peptides regions, two within the VP1u sequence, 17–28 and 113–124, and six within the common VP3 sequence, 241–260, 305–356, 401–420, 443–460, 473–484, and 697–716 (**Table 1**) (62). As already stated, there is no structure available for the VP1u. The VP3 sequences are localized to βB, strands βD-βE and the intervening loop that forms the 5-fold channel including VR-II, strands βF-βG, the outer finger of the 3-fold protrusion that contains VR-IV, a strand region of the GH loop, and the 2/5-fold wall that contains VR-IX, respectively, in the

VP3 crystal structure. The peptides containing residues 305–356, 401–420, 443–460 were considered the core neutralizing peptides. A number of these peptides are located in VP regions that are inside the capsids, and thus their ability to block capsid binding by human serum likely reflects the polyclonal nature of the human serum, which must include antibody responses to denatured VP regions or fragments.

Wobus et al. (55) also used a peptide mapping strategy to determine the epitopes of three mouse MAbs, A1, A69, and B1 previously reported to react against AAV2 (72, 73). An AAV2 *cap* gene fragment phage display library was screened with the antibodies and positive clones were scanned for binding by the antibodies, as overlapping 15-mer peptides on a membrane, and further confirmed by peptide competition in a Western blot. Linear epitopes were mapped for A1 (residues 123–131) within VP1u, A69 (residues 171–182) in theVP1/VP2 region and for B1 (residues 726–733) at the C-terminus of VP3. Consistently, these antibodies react against denatured capsids. The epitopes for three conformational AAV2 directed antibodies, A20, C37-B, and D3 (55) were also identified through a similar strategy. Overlapping 10-mer peptides (covering the AAV2 capsid sequence) detected by the antibodies were confirmed by an ELISA assay. Multiple peptides were identified for these antibodies consistent with their recognition of the assembled capsid, although C37-B and D3 were also capable of recognizing the VPs (55). The epitopes proposed for the three MAbs were: 272–281, 369–378, and 566–575 for A20; 492–503 and 601–610 for C37–B; and 474–483 for D3 (**Table 2**). The A20 epitope residues are mostly located below surface loops containing VR-I (for 272–281), the HI loop (for 369–378), and VR-V (for 566–575); the C37-B epitope peptides are located in VR-V (492– 503) and buried at the 3-fold axis (601–610), and the D3 epitope is buried close to the 3-fold axis (474–483) (**Figure 2B**). As previously mentioned, the A20 MAb neutralizes virus infection at a post cell entry step and the C37-B antibody inhibits HSPG receptor attachment by AAV2. The D3 antibody is non-neutralizing, which is consistent with the mostly buried location of its mapped epitope.

#### **PEPTIDE INSERTION**

Wobus et al. (55) attempted to further confirm the binding sites for the A20, C37-B, and D3 antibodies as well as another antibody named C24-B using mutant AAV2 capsids onto which a 14 amino acid integrin binding ligand, L14, had been inserted. Prior to the availability of the AAV2 capsid structure, Girod et al. (74) utilized the 3D structure of canine parvovirus (75) to generate a 3D homology model for AAV2, which was used for predicting potential capsid surface sites onto which this peptide could be inserted for re-targeting AAV2 (74). Six such sites, 261, 381, 447, 534, 573, and 587, were identified. Testing of the antibody binding properties of these insertion mutants with the A20, C37- B, D3, and C24-B antibodies showed that the insertions at 261, 381, 534, and 573 decreased A20 binding; at 534, 573, and 587 decreased C37-B binding; at 261, 381, 534, and 573 decreased D3 binding; and at 534, 573, and 587 decreased C24-B binding (**Table 2**). These insertion points did not overlap with the epitopes predicted based on peptide scanning data, except for residue 573 for A20 (55). For the A20 and C37-B MAbs, a number of

the sites do overlap with or are close to epitope regions mapped using site-directed mutagenesis and structural biology as discussed below.

Huttner et al. tested the ability of these six insertion mutants to evade binding by human serum samples in an ELISA assay (63). The A20 and C37-B antibodies were used as positive controls. Insertion mutations at amino acid positions 534 and 573 reduced human anti-sera binding, in 19/29 tested samples, by up to 30% compared to the wild-type AAV2. Both of these residues are buried inside the protrusions surround the 3-fold axis. Thus their negative impact on serum binding is likely due to disruption of the surface loops that assembles the protrusions suggested above to be important for antigenic reactivity for the AAVs. The mutant with an insertion at position 587 only slightly impaired serum binding in ELISA assays, but was able to transduce Hela cells in the presence of human serum. The inserted L14 ligand also enabled this mutant to infect B16F10 cells, a cell line which is nonpermissive to the parental AAV2, in the presence of human serum. These observations are consistent with the fact that residue 587 of AAV2 is located in a capsid region involved in immunogenicity, e.g., the C37-B epitope, and cellular attachment, e.g., proximity to the HSPG binding site (65, 66).

#### **SITE-DIRECTED MUTAGENESIS**

The best example of the use of site-directed mutagenesis for antigenic epitope mapping is provided by the study by Lochrie et al. (38). This group used the crystal structure of AAV2 (32) to identify sites for mutations on the capsid surface at amino acid positions predicted to be potential antigenic sites based on the docking of a murine IgG2a. All the mutants (57 alanine substitutions, 41 non-alanine substitutions) were screened for binding and neutralization ability with A20, three individual human serum samples, and IVIG. The mutated positions that decreased neutralization by A20 were 263, 264, 384, 385, 548, and 708. These residues are different to the amino acid stretches mapped by Wobus et al. (55) and Hunter et al. (63), but are located structurally proximate to these residues and are all clustered on the 2/5-fold wall (**Table 2** and **Figure 1**). The three human sera and IVIG screening identified several epitopes (**Table 1**) due to the complexity of the polyclonal response being tested. Different mutants showed resistance to different serum, and the mutations that resulted in neutralization escape from the individual serum and IVIG were located over a capsid region that was three times larger than an average Fab epitope footprint and spanned the 3-fold protrusions as well as the 2/5-fold wall. In addition, the ability to escape from human sera