AAV Immunogenicity: A Matter of Sensitivity
Authors: Federico Mingozzi
Journal: Molecular Therapy (2018)

Gene transfer with adeno-associated virus (AAV) vectors has yielded clinically relevant results in several preclinical and clinical studies, with several candidate treatments proceeding to late-stage clinical trials and two AAV-based gene therapy drugs already approved. Despite the excitement, immunogenicity of AAV vectors remains an important hurdle associated with the technology,1Mingozzi F. High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.Annu. Rev. Virol. 2017; 4: 511-534Crossref PubMed Scopus (118) Google Scholar both in preclinical and clinical studies. Because humans and non-human primates (NHPs) are exposed to wild-type AAV, they often carry anti-capsid neutralizing antibodies (NAbs) that can cross-react with several of the different serotypes used for gene transfer.1Mingozzi F. High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.Annu. Rev. Virol. 2017; 4: 511-534Crossref PubMed Scopus (118) Google Scholar Furthermore, upon vector administration, anti-AAV NAbs quickly develop at high titers and persist for several years,1Mingozzi F. High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.Annu. Rev. Virol. 2017; 4: 511-534Crossref PubMed Scopus (118) Google Scholar thus preventing vector re-administration. Depending on various factors, including the route of administration and the vector dose and formulation, susceptibility of AAV vectors to NAbs can vary.1Mingozzi F. High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.Annu. Rev. Virol. 2017; 4: 511-534Crossref PubMed Scopus (118) Google Scholar For example, in NHPs, doses of 5 × 1012 vector genomes (vg)/kg of AAV8 vectors injected via the hepatic artery were completely neutralized by AAV NAb titers as low as 1:5.2Jiang H. Couto L.B. Patarroyo-White S. Liu T. Nagy D. Vargas J.A. Zhou S. Scallan C.D. Sommer J. Vijay S. et al.Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.Blood. 2006; 108: 3321-3328Crossref PubMed Scopus (248) Google Scholar This is consistent with findings in mice and humans, showing that even low NAb titers can completely block liver transduction of AAV vectors delivered into the bloodstream.1Mingozzi F. High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.Annu. Rev. Virol. 2017; 4: 511-534Crossref PubMed Scopus (118) Google Scholar The clear definition of the baseline immunological parameters of gene transfer is therefore important to ensure accurate results interpretation in both preclinical and clinical studies. A recent series of four reports describing the treatment of NHP models of non-alcoholic steatohepatitis with AAV8 vectors represents an example of the issue.3Ji Y.X. Huang Z. Yang X. Wang X. Zhao L.P. Wang P.X. Zhang X.J. Alves-Bezerra M. Cai L. Zhang P. et al.The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis.Nat. Med. 2018; 24: 213-223Crossref PubMed Scopus (84) Google Scholar, 4Zhang P. Wang P.X. Zhao L.P. Zhang X. Ji Y.X. Zhang X.J. Fang C. Lu Y.X. Yang X. Gao M.M. et al.The deubiquitinating enzyme TNFAIP3 mediates inactivation of hepatic ASK1 and ameliorates nonalcoholic steatohepatitis.Nat. Med. 2018; 24: 84-94Crossref PubMed Scopus (123) Google Scholar, 5Zhao G.N. Zhang P. Gong J. Zhang X.J. Wang P.X. Yin M. Jiang Z. Shen L.J. Ji Y.X. Tong J. et al.Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4.Nat. Med. 2017; 23: 742-752Crossref PubMed Scopus (100) Google Scholar, 6Wang P.X. Ji Y.X. Zhang X.J. Zhao L.P. Yan Z.Z. Zhang P. Shen L.J. Yang X. Fang J. Tian S. et al.Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.Nat. Med. 2017; 23: 439-449Crossref PubMed Scopus (146) Google Scholar The animals had been selected for being negative or having very low anti-AAV8 NAb titers,7Wang P.X. Zhao G.N. Ji Y.X. Zhang P. Zhang X.J. Gong J. Zhao L.P. Yan Z.Z. Yin M. Jiang Z. et al.Wang et al. reply.Nat. Med. 2018; 24: 700-701Crossref PubMed Scopus (2) Google Scholar and 2 × 1013 vg of vector (roughly equivalent to 5 × 1012 vg/kg2Jiang H. Couto L.B. Patarroyo-White S. Liu T. Nagy D. Vargas J.A. Zhou S. Scallan C.D. Sommer J. Vijay S. et al.Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.Blood. 2006; 108: 3321-3328Crossref PubMed Scopus (248) Google Scholar) were administered intravascularly via the portal vein. The assay used to determine NAb titers was based on a standard protocol, similar to the one used in other NHP studies,2Jiang H. Couto L.B. Patarroyo-White S. Liu T. Nagy D. Vargas J.A. Zhou S. Scallan C.D. Sommer J. Vijay S. et al.Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.Blood. 2006; 108: 3321-3328Crossref PubMed Scopus (248) Google Scholar but with a cutoff for positivity of 1:10.7Wang P.X. Zhao G.N. Ji Y.X. Zhang P. Zhang X.J. Gong J. Zhao L.P. Yan Z.Z. Yin M. Jiang Z. et al.Wang et al. reply.Nat. Med. 2018; 24: 700-701Crossref PubMed Scopus (2) Google Scholar In the original publications,3Ji Y.X. Huang Z. Yang X. Wang X. Zhao L.P. Wang P.X. Zhang X.J. Alves-Bezerra M. Cai L. Zhang P. et al.The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis.Nat. Med. 2018; 24: 213-223Crossref PubMed Scopus (84) Google Scholar, 4Zhang P. Wang P.X. Zhao L.P. Zhang X. Ji Y.X. Zhang X.J. Fang C. Lu Y.X. Yang X. Gao M.M. et al.The deubiquitinating enzyme TNFAIP3 mediates inactivation of hepatic ASK1 and ameliorates nonalcoholic steatohepatitis.Nat. Med. 2018; 24: 84-94Crossref PubMed Scopus (123) Google Scholar, 5Zhao G.N. Zhang P. Gong J. Zhang X.J. Wang P.X. Yin M. Jiang Z. Shen L.J. Ji Y.X. Tong J. et al.Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4.Nat. Med. 2017; 23: 742-752Crossref PubMed Scopus (100) Google Scholar, 6Wang P.X. Ji Y.X. Zhang X.J. Zhao L.P. Yan Z.Z. Zhang P. Shen L.J. Yang X. Fang J. Tian S. et al.Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.Nat. Med. 2017; 23: 439-449Crossref PubMed Scopus (146) Google Scholar the authors failed to describe the methodology used to screen the animals for anti-AAV NAbs, which generated some controversy8Xiao W. Gao G. Ling C. Herzog R.W. Xiao X. Samulski R.J. Impact of neutralizing antibodies against AAV is a key consideration in gene transfer to nonhuman primates.Nat. Med. 2018; 24: 699Crossref PubMed Scopus (8) Google Scholar related to the selection of animals for inclusion in the study and, therefore, associated with the reliability of the results described. Additionally, in one of the publications,6Wang P.X. Ji Y.X. Zhang X.J. Zhao L.P. Yan Z.Z. Zhang P. Shen L.J. Yang X. Fang J. Tian S. et al.Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.Nat. Med. 2017; 23: 439-449Crossref PubMed Scopus (146) Google Scholar the therapeutic vector was administered twice, at day 0 and week 7, without taking into account that the second vector infusion likely did not result in additional liver transduction due to the likely development of high-titer NAbs. These points were more recently clarified by the authors,7Wang P.X. Zhao G.N. Ji Y.X. Zhang P. Zhang X.J. Gong J. Zhao L.P. Yan Z.Z. Yin M. Jiang Z. et al.Wang et al. reply.Nat. Med. 2018; 24: 700-701Crossref PubMed Scopus (2) Google Scholar who argued that animals were carefully prescreened for anti-AAV8 NAbs and that the results of the screening were not shown because the authors "did not feel compelled to report those data." While this can be a reasonable statement, given the relevance of the NAb assay readout on the efficacy of gene transfer, it is recommended to specify both the methodology used to prescreen animals and the actual NAb titers measured. Furthermore, it seems that the cutoff chosen for inclusion of the animals in the study, 1:10, was higher than what represents a seronegative animal, although no details on the sensitivity of the assay used for screening were provided.2Jiang H. Couto L.B. Patarroyo-White S. Liu T. Nagy D. Vargas J.A. Zhou S. Scallan C.D. Sommer J. Vijay S. et al.Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.Blood. 2006; 108: 3321-3328Crossref PubMed Scopus (248) Google Scholar Finally, the authors did not report the NAb titers measured at week 7, at the time of vector re-administration,6Wang P.X. Ji Y.X. Zhang X.J. Zhao L.P. Yan Z.Z. Zhang P. Shen L.J. Yang X. Fang J. Tian S. et al.Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.Nat. Med. 2017; 23: 439-449Crossref PubMed Scopus (146) Google Scholar despite the fact that, in this case, NAb titers would be extremely useful to understand whether vector redosing was successful. The lesson is clear: translating gene transfer approaches from small to large animals and to humans, should prompt investigators to take immune responses to AAV vectors into careful account. This consideration is not restricted to capsid—it also concerns the use of reporter transgenes like GFP, which are tolerated in mice but can be highly immunogenic in primates.9Gao G. Wang Q. Calcedo R. Mays L. Bell P. Wang L. Vandenberghe L.H. Grant R. Sanmiguel J. Furth E.E. Wilson J.M. Adeno-associated virus-mediated gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses.Hum. Gene Ther. 2009; 20: 930-942Crossref PubMed Scopus (70) Google Scholar For example, the lack of any immunogenicity assessment in two of the NHP studies described above,3Ji Y.X. Huang Z. Yang X. Wang X. Zhao L.P. Wang P.X. Zhang X.J. Alves-Bezerra M. Cai L. Zhang P. et al.The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis.Nat. Med. 2018; 24: 213-223Crossref PubMed Scopus (84) Google Scholar, 5Zhao G.N. Zhang P. Gong J. Zhang X.J. Wang P.X. Yin M. Jiang Z. Shen L.J. Ji Y.X. Tong J. et al.Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4.Nat. Med. 2017; 23: 742-752Crossref PubMed Scopus (100) Google Scholar in which the therapeutic transgene was co-expressed with GFP separated by a T2A sequence,3Ji Y.X. Huang Z. Yang X. Wang X. Zhao L.P. Wang P.X. Zhang X.J. Alves-Bezerra M. Cai L. Zhang P. et al.The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis.Nat. Med. 2018; 24: 213-223Crossref PubMed Scopus (84) Google Scholar, 5Zhao G.N. Zhang P. Gong J. Zhang X.J. Wang P.X. Yin M. Jiang Z. Shen L.J. Ji Y.X. Tong J. et al.Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4.Nat. Med. 2017; 23: 742-752Crossref PubMed Scopus (100) Google Scholar raises concerns regarding the kinetics of transgene expression over time and its durability. Since the initial reports of capsid immune responses,1Mingozzi F. High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.Annu. Rev. Virol. 2017; 4: 511-534Crossref PubMed Scopus (118) Google Scholar our understanding of the impact of AAV immunogenicity has evolved considerably. We now have several tools that can be used for routine immunomonitoring of immune responses in AAV gene transfer. A lot more needs to be done, in particular, on the front of standardization of these crucially important assays, like the ones used to determine anti-AAV NAbs, to ensure the same level of sensitivity and reproducibility across studies and thus allowing a more straightforward comparison of results. Nevertheless, capturing the knowledge of vector-host immune system interactions into the design of preclinical and clinical studies should be the goal for the entire scientific community. F.M. is an employee of Spark Therapeutics, Inc. The author gratefully acknowledges support from EU grant EU-FP7 ERC-Co-G 617432 MoMAAV.