Prevalence of Pre-Existing Neutralizing Antibodies Against Adeno-Associated Virus Serotypes 1, 2, 5, 6, 8, and 9 in Sera of Different Pig Strains
Abstract
Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5–6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes.
These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were <3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.
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References
1. Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 2019;18:358–378.
2. Bulaklak K, Xiao X. Therapeutic advances in musculoskeletal AAV targeting approaches. Curr Opin Pharmacol 2017;34:56–63.
3. Gao GP, Alvira MR, Wang L, et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 2002;99:11854–11859.
4. Monahan PE, Négrier C, Tarantino M, et al. Emerging immunogenicity and genotoxicity considerations of adeno-associated virus vector gene therapy for hemophilia. J Clin Med 2021;10:2471.
5. Bowles DE, McPhee SW, Li C, et al. Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther 2012;20:443–455.
6. Weber T. Anti-AAV Antibodies in AAV gene therapy: current challenges and possible solutions. Front Immunol 2021;12:658399.
7. Gorovits B, Fiscella M, Havert M, et al. Recommendations for the development of cell-based anti-viral vector neutralizing antibody assays. AAPS J 2020;22:24.
8. Kotterman MA, Yin L, Strazzeri JM, et al. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther 2015;22:116–126.
9. Majowicz A, Nijmeijer B, Lampen MH, et al. Therapeutic hFIX activity achieved after single AAV5-hFIX treatment in hemophilia B patients and NHPs with pre-existing anti-AAV5 NABs. Mol Ther Methods Clin Dev 2019;14:27–36.
10. Chirmule N, Propert K, Magosin S, et al. Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther 1999;6:1574–1583.
11. Erles K, Sebokova P, Schlehofer JR. Update on the prevalence of serum antibodies (IgG and IgM) to adeno-associated virus (AAV). J Med Virol 1999;59:406–411.
12. Halbert CL, Miller AD, McNamara S, et al. Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: implications for gene therapy using AAV vectors. Hum Gene Ther 2006;17:440–447.
13. Calcedo R, Vandenberghe LH, Gao G, et al. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009;199:381–390.
14. Calcedo R, Franco J, Qin Q, et al. Preexisting neutralizing antibodies to adeno-associated virus capsids in large animals other than monkeys may confound in vivo gene therapy studies. Hum Gene Ther Methods 2015;26:103–105.
15. Wang L, Calcedo R, Bell P, et al. Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. Hum Gene Ther 2011;22:1389–1401.
16. Boutin S, Monteilhet V, Veron P, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther 2010;21:704–712.
17. Rapti K, Louis-Jeune V, Kohlbrenner E, et al. Neutralizing antibodies against AAV serotypes 1, 2, 6, and 9 in sera of commonly used animal models. Mol Ther 2012;20:73–83.
18. Wang D, Zhong L, Li M, et al. Adeno-associated virus neutralizing antibodies in large animals and their impact on brain intraparenchymal gene transfer. Mol Ther Methods Clin Dev 2018;11:65–72.
19. Swindle MM, Makin A, Herron AJ, et al. Swine as models in biomedical research and toxicology testing. Vet Pathol 2012;49:344–356.
20. Lelovas PP, Kostomitsopoulos NG, Xanthos TT. A comparative anatomic and physiologic overview of the porcine heart. J Am Assoc Lab Anim Sci 2014;53:432–438.
21. Fish KM, Ladage D, Kawase Y, et al. AAV9.I-1c delivered via direct coronary infusion in a porcine model of heart failure improves contractility and mitigates adverse remodeling. Circ Heart Fail 2013;6:310–317.
22. Ishikawa K, Fish KM, Tilemann L, et al. Cardiac I-1c overexpression with reengineered AAV improves cardiac function in swine ischemic heart failure. Mol Ther 2014;22:2038–2045.
23. Raake PW, Hinkel R, Müller S, et al. Cardio-specific long-term gene expression in a porcine model after selective pressure-regulated retroinfusion of adeno-associated viral (AAV) vectors. Gene Ther 2008;15:12–17.
24. Hulsegge I, Calus M, Hoving-Bolink R, et al. Impact of merging commercial breeding lines on the genetic diversity of Landrace pigs. Genet Sel Evol 2019;51:60.
25. Tseng YS, Vliet KV, Rao L, et al. Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies. J Virol Methods 2016;236:105–110.
26. Tseng Y-S, Gurda B, Chipman P, et al. Adeno-associated virus serotype 1 (AAV1)- and AAV5-antibody complex structures reveal evolutionary commonalities in parvovirus antigenic reactivity. J Virol 2015;89:1794–1808.
27. Sonntag F, Köther K, Schmidt K, et al. The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes. J Virol 2011;85:12686–12697.
28. Kavita U, Dai Y, Salvador L, et al. Development of a chemiluminescent ELISA method for the detection of total anti-adeno associated virus serotype 9 (AAV9) antibodies. Hum Gene Ther Methods 2018;29:237–250.
29. Rogers GL, Huang C, Clark R, et al. Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes. bioRxiv 2021:2021.05.03.440656.
30. Shen S, Bryant KD, Brown SM, et al. Terminal N-linked galactose is the primary receptor for adeno-associated virus 9. J Biol Chem 2011;286:13532–13540.
31. Calcedo R, Morizono H, Wang L, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol 2011;18:1586–1588.
32. Perocheau DP, Cunningham S, Lee J, et al. Age-related seroprevalence of antibodies against AAV-LK03 in a UK population cohort. Hum Gene Ther 2019;30:79–87.
33. Fitzpatrick Z, Leborgne C, Barbon E, et al. Influence of pre-existing anti-capsid neutralizing and binding antibodies on AAV vector transduction. Mol Ther Methods Clin Dev 2018;9:119–129.
34. Kruzik A, Koppensteiner H, Fetahagic D, et al. Detection of biologically relevant low-titer neutralizing antibodies against adeno-associated virus require sensitive in vitro assays. Hum Gene Ther Methods 2019;30:35–43.
35. Li P, Boenzli E, Hofmann-Lehmann R, et al. Pre-existing antibodies to candidate gene therapy vectors (adeno-associated vector serotypes) in domestic cats. PLoS One 2019;14:e0212811.
36. Bello A, Chand A, Aviles J, et al. Novel adeno-associated viruses derived from pig tissues transduce most major organs in mice. Sci Rep 2014;4:6644.
37. Bello A, Tran K, Chand A, et al. Isolation and evaluation of novel adeno-associated virus sequences from porcine tissues. Gene Ther 2009;16:1320–1328.
38. Ellis BL, Hirsch ML, Barker JC, et al. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1–AAV9) and one engineered adeno-associated virus serotype. Virol J 2013;10:74-.
39. Krotova K, Aslanidi G. Modifiers of adeno-associated virus-mediated gene expression in implication for serotype-universal neutralizing antibody assay. Hum Gene Ther 2020;31:1124–1131.
40. Guo P, Zhang J, Chrzanowski M, et al. Rapid AAV-neutralizing antibody determination with a cell-binding assay. Mol Ther Methods Clin Dev 2019;13:40–46.
41. Gurda BL, Raupp C, Popa-Wagner R, et al. Mapping a neutralizing epitope onto the capsid of adeno-associated virus serotype 8. J Virol 2012;86:7739–7751.
42. Clauss S, Bleyer C, Schüttler D, et al. Animal models of arrhythmia: classic electrophysiology to genetically modified large animals. Nat Rev Cardiol 2019;16:457–475.
43. Hryhorowicz M, Zeyland J, Słomski R, et al. Genetically modified pigs as organ donors for xenotransplantation. Mol Biotechnol 2017;59:435–444.
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Published online: 19 April 2022
Published in print: April 2022
Published ahead of production: 16 December 2021
Accepted: 30 November 2021
Received: 20 August 2021
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