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. 2012 Mar;23(3):287-94.
doi: 10.1089/hum.2011.125. Epub 2012 Jan 11.

Humoral immunity to AAV-6, 8, and 9 in normal and dystrophic dogs

Jin-Hong Shin  1 , Yongping Yue, Bruce Smith, Dongsheng Duan
Affiliations

Humoral immunity to AAV-6, 8, and 9 in normal and dystrophic dogs

Jin-Hong Shin et al. Hum Gene Ther. 2012 Mar.

Abstract

Adeno-associated virus (AAV)-6, 8, and 9 are promising gene-delivery vectors for testing novel Duchenne muscular dystrophy gene therapy in the canine model. Humoral immunity greatly influences in vivo AAV transduction. However, neutralizing antibodies to AAV-6, 8, and 9 have not been systemically examined in normal and dystrophic dogs. To gain information on the seroprevalence of antibodies to AAV-6, 8, and 9, we measured neutralizing antibody titers using an in vitro transduction inhibition assay. We examined 72 naive serum samples and 26 serum samples obtained from dogs that had received AAV gene transfer. Our data demonstrated that AAV-6 neutralizing antibody was the most prevalent antibody in dogs irrespective of age, gender, disease status (dystrophic or not), and prior parvovirus vaccination history. Surprisingly, high-level anti-AAV-6 antibody was detected at birth in newborn puppies. Further, a robust antibody response was induced in affected, but not normal newborn dogs following systemic AAV gene transfer. Taken together, our data have provided an important baseline on the seroprevalence of AAV-6, 8, and 9 neutralizing antibodies in normal and Duchenne muscular dystrophy dogs. These results will help guide translational AAV gene-therapy studies in dog models of muscular dystrophy.

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Figures

FIG. 1.
FIG. 1.
Preexisting AAV-6, 8, and 9 NAbs in naive dogs. (A–C) The NAb titer at different ages in normal, affected, and carrier dogs. Please note that the sample size is not reflected in the graphs. In many cases, results from several dogs of the same age were identical. (D) Correlation of disease status and seroprevalence. For affected and normal groups, data from male and female dogs were pooled. Affected males carry dystrophin gene mutation in the X chromosome. Affected females carry dystrophin gene mutation in both X chromosomes. Normal dogs do not carry dystrophin gene mutation. The carrier group consisted of only female dogs; they have one normal X chromosome and one dystrophin gene-mutated X chromosome. n=32 for normal dogs; n=21 for affected dogs; n=19 for carrier dogs. *Anti-AAV-6 antibody titers are significantly different from those of anti-AAV-8 and anti-AAV-9 antibody titers in the same category (normal, carrier, or affected).
FIG. 2.
FIG. 2.
Parvovirus vaccination and gender do not influence AAV NAb titer. (A) Correlation of parvovirus vaccination and NAbs to AAV-6, 8, and 9. n=56 for unvaccinated dogs; n=16 for vaccinated dogs. *Anti-AAV-6 antibody titer in unvaccinated normal dogs is significantly higher than that of vaccinated normal dogs by one-way ANOVA. (B) Comparison of seroprevalence to AAV-6, 8, and 9 between male and female dogs. n=36 for male; n=36 for female.
FIG. 3.
FIG. 3.
Characterization of the AAV-6 NAb. (A) Correlation of NAbs between dams and newborn puppies (this panel also includes data for AAV-8 and AAV-9 antibodies). *Significantly different between dams and puppies by t test. n=3 for dams; n=23 for puppies. (B) Kinetics of the AAV-6 NAb as dogs age. (C) AAV-6 NAb titer in serum samples collected at different months. None of the serum sample was collected between October and December.
FIG. 4.
FIG. 4.
Development of NAb following intravascular injection of Y731F AAV-9 in newborn dogs. (A) Two normal puppies and five affected puppies received systemic Y731F AAV-9 tyrosine mutant within 2 hr after birth. The NAb titer was determined at 3–7 weeks of age. Asterisk, significantly different from that of normal dogs by t test. (B) The time course of AAV-9 NAb development in five affected puppies that received neonatal intravenous Y731F AAV-9 tyrosine mutant injection.
FIG. 5.
FIG. 5.
Humoral response to local muscle AAV injection in adult dogs. (A) Induction of the NAb to AAV-9 following intramuscular (im) injection of Y731F tyrosine mutant AAV-9 in two normal dogs. One dog received transient immune suppression, and the other dog did not. (B) Induction of the NAb to AAV-6 following intramuscular injection of Y445F tyrosine mutant AAV-6 in two affected dogs. Both dogs received transient immune suppression.
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