Daniel J. Hui

CD8 + T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediatedgene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid–specific memory CD8+ T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid–specific CD8+ T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.

AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.

In a clinical trial for adeno-associated virus serotype 1 (AAV-1)-mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-gamma enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4(+) and CD8(+) T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti-AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.

Overcoming Preexisting Humoral Immunity to AAV Using Capsid Decoys

Capsid decoys enhance the efficacy of AAV vector transduction after systemic delivery in the presence of neutralizing antibodies. A Slight of Hand for Gene Therapy Gene therapy has been quite successful—in animal models. But when it comes to translating gene therapy to humans, there have only been a few shining successes. One limiting factor has been the vectors used. Adeno-associated virus (AAV) vectors are safe, noninvasive, and potentially effective; however, people who have been previously exposed to AAV have preexisting neutralizing antibodies that block gene delivery. Now, Mingozzi et al. trick these antibodies into binding empty viral capsid, overcoming their inhibitory effects. The authors hypothesized that introducing empty capsids along with the gene therapy vector would titrate out the neutralizing antibody response to AAV, allowing for successful gene therapy even in the presence of preexisting neutralizing antibodies. They found that varying the ratio of empty capsid to gene therapy vector could successfully inhibit the neutralizing antibody response in both human serum and a mouse model by serving as a decoy for antibody binding. The authors then mutated the receptor binding site of their capsid so that it could bind the neutralizing antibody but not target cells, further increasing the safety profile of this approach. These capsid decoys worked in a dose-dependent manner and were successful even with high antibody titers. What’s more, they were safe and effective in rhesus macaques. Although this approach remains to be tested in humans, tricking neutralizing antibodies with decoys may be the next step in advancing gene therapy in the clinic. Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient’s anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid-derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.

Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector–mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4+FoxP3+IL-10+ T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle.

Diverse IgG Subclass Responses to Adeno-associated Virus Infection and Vector Administration

Humoral immune responses occur following exposure to Adeno‐associated virus (AAV) or AAV vectors. Many studies characterized antibody responses to AAV, but human IgG subclass responses to AAV have not been previously described. In this study, IgG subclass responses were examined in serum samples of normal human subjects exposed to wild‐type AAV, subjects injected intramuscularly with AAV vectors and subjects injected intravascularly with AAV vectors. A diversity of IgG subclass responses to AAV capsid were found in different subjects. IgG1 was found to be the dominant response. IgG2, IgG3, and IgG4 responses were also observed in most normal human subjects; IgG2 and IgG3 each represented the major fraction of total anti‐AAV capsid IgG in a subset of normal donors. Subjects exposed to AAV vectors showed IgG responses to AAV capsid of all four IgG subclasses. IgG responses to AAV capsid in clinical trial subjects were inversely proportional to the level of pre‐existing anti‐AAV antibody and independent of the vector dose. The high levels of anti‐AAV capsid IgG1 can mask differences in IgG2, IgG3, and IgG4 responses that were observed in this study. Analysis of IgG subclass distribution of anti‐AAV capsid antibodies indicates a complex, non‐uniform pattern of responses to this viral antigen. J. Med. Virol. 81:65–74, 2009.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

Enhanced T Cell Function in a Mouse Model of Human Glycosylation

Clinical evidence for a more active immune response in humans compared with our closest hominid relative, the chimpanzee, includes the progression of HIV infection to AIDS, hepatitis B– and C–related inflammation, autoimmunity, and unwanted harmful immune responses to viral gene transfer vectors. Humans have a unique mutation of the enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH), causing loss of expression of the sialic acid Neu5Gc. This mutation, occurring 2 million years ago, likely altered the expression and function of ITIM-bearing inhibitory receptors (Siglecs) that bind sialic acids. Previous work showed that human T cells proliferate faster than chimpanzee T cells upon equivalent stimulation. In this article, we report that Cmah−/− mouse T cells proliferate faster and have greater expression of activation markers than wild-type mouse T cells. Metabolically reintroducing Neu5Gc diminishes the proliferation and activation of both human and murine Cmah−/− T cells. Importantly, Cmah−/− mice mount greater T cell responses to an adenovirus encoding an adeno-associated virus capsid transgene. Upon lymphocytic choriomeningitis virus infection, Cmah−/− mice make more lymphocytic choriomeningitis virus–specific T cells than WT mice, and these T cells are more polyfunctional. Therefore, a uniquely human glycosylation mutation, modeled in mice, leads to a more proliferative and active T cell population. These findings in a human-like mouse model have implications for understanding the hyperimmune responses that characterize some human diseases.

185. Safety Study by Validated Immunoassays in a Phase III Study of Subjects with Inherited Retinal Dystrophy Due to Mutations in the Gene Encoding Human Retinal Pigment Epithelium-Specific Protein 65 (RPE65) Injected with Adeno-Associated Viral Vectors

Mutations in the gene encoding Retinal Pigment Epithelium-Specific Protein 65 (RPE65) cause impaired vision from an early age and eventually total vision loss later in life. Recent work by our group and others demonstrated the use of SPK-RPE65, an adeno-associated viral (AAV) vector, to deliver the gene stably in small and large animal models, while Phase 1 and Phase 3 clinical trials with SPK-RPE65 showed promising results (Maguire et al., 2008; Maguire et al., 2009; Simonelli et al., 2010; Maguire, AAO 2015). The Phase 3 trial was an open-label, randomized study using a subretinally-delivered AAV2 vector to augment the RPE65 gene. Thirty one subjects were enrolled from centers at The Childrens Hospital of Philadelphia or University of Iowa. Twenty of the 21 intervention group subjects received 1.5E11 vg non-simultaneous injections to each eye. In addition to a separate efficacy assessment that measured the primary and secondary endpoints for the trial, an independent safety study was performed that included previously established immunological assays designed to monitor cellular immune responses. Two assays were subjected to further validation in-house and used for immune analysis of clinical samples obtained from all intervention subjects. An Enzyme-Linked Immunosorbent Assay (ELISA), capable of detecting a titer of at least 1.55 ug/mL anti-AAV2-capsid human IgG, was performed on 21 subjects covering up to 4 timepoints (baseline prior to injection, day 30, day 90 and 1 year). To better evaluate the results, the subjects were placed into 6 categories based on their antibody titer profile (Table 1Table 1).Table 1Anti-AAV2 Profile of Intervention Group (n=21)Number of SubjectsAnti AAV Titer Range (ug/mL)Anti-AAV2 Profile7<1.55Below quantification limit31.72 – 2.91Low pre-existing antibody titer216.37 – 19.61Moderate pre-existing antibody titer454.39 – 248.55High pre-existing antibody titer41.73 – 87.02Antibody titer developed after vector administration1N/AWithdrew View Table in HTML The other immunoassay performed on the intervention group was an interferon-γ Enzyme-Linked Immunospot Assay (ELISPOT) to assess T cell responses against AAV2 capsid or RPE65 transgene product. The same set of intervention subjects/timepoints was analyzed with positive T cell responses defined as ≥50 spot forming units (SFU) and 3-fold the background (media) control for AAV2 and greater than the statistically determined cutoff of 161.3 SFU for RPE65. Eighteen of the 21 intervention subjects tested negative for T cell responses against AAV2 and RPE65 across all timepoints. One subject was positive against AAV2 capsid at baseline (55.0 SFU) and positive against RPE65 at the 1 year timepoint (171.7 SFU). Another subject was positive at 1 year for RPE65 only (170.0 SFU). These positive responses were considered very weak with respect to threshold cutoff values. One subject displayed a moderate response (518.3 SFU) against RPE65 at baseline only, while all subsequent timepoints were negative. The positive T cell responses observed at baseline prior to vector administration are unlikely to be related to gene transfer. Considering the cumulative immune response data collected from the two validated immunoassays performed here, the Phase 3 trial provided results supporting the immunologic tolerability of the delivery of AAV2 vector to the subretinal space in the eye to treat inherited retinal dystrophy due to mutations in RPE65. Efforts are ongoing to correlate these findings with available efficacy data.