Siders WM, Shields J, Kaplan J, Lukason M, Woodworth L, et al.
Human gene therapy •
The use of adeno-associated viral (AAV) vectors for gene replacement therapy is currently being explored in several clinical indications. However, reports have suggested that input capsid proteins from AAV-2 vector particles may result in the stimulation of cytotoxic T lymphocyte (CTL) responses that can result in a loss of transduced cells. To explore the impact of anti-AAV CTLs on AAV-mediated transgene expression, both immunocompetent C57BL=6 mice and B cell-deficient muMT mice were immunized against the AAV2 capsid protein (Cap) and were injected intravenously with an AAV-2 vector encoding alpha-galactosidase (alpha-Gal). C57BL=6 mice, which developed both CTL and neutralizing antibody responses against Cap, failed to show any detectable alpha-Gal expression. In contrast, serum alpha-Gal levels comparable to those of naive mice were observed in muMT mice despite the presence of robust CTL activity against Cap, indicating that preexisting Cap-specific CTLs did not have any effect on the magnitude and duration of transgene expression. The same strategy was used to assess the impact of CTLs against the alpha-Gal transgene product on AAV-mediated gene delivery and persistence of transgene expression. Preimmunization of muMT mice with an Ad=alpha-Gal vector induced a robust CTL response to alpha-Gal. When these mice were injected with AAV2=alpha-Gal vector, initial levels of alpha-Gal expression were reduced by more than 1 log and became undetectable by 2 weeks postinjection. Overall, our results indicate that CTLs against the transgene product as opposed to AAV capsid protein are more likely to interfere with AAV transgene expression.
Specific anti-Salmonella enteritidis (SE) and natural anti-alpha-gal epitope (Galalpha1-3Galbeta-1-4GlcNAc-R; anti-Gal) antibodies were measured in plasma sample pools and individual bile specimens obtained from hens differing in diet efficiency. More SE somatic (O) and flagellar (H) antibodies were found in plasma pools from efficient hens (R-) compared with nonefficient hens (R+) after oral challenge with live SE. Mean titers of somatic agglutinins in bile were 2.3 in R- hens and 1.9 in R+ hens (P = 0.06) following live challenge. Salmonella enteritidis antibodies were also found in bile of nonchallenged hens of both types but their levels were not significantly different. Flagellar (H) agglutinin scores were higher in SE-challenged hens compared with nonchallenged hens (3.1 vs. 2.1; P < or = 0.004) but efficiency types did not differ. Bile also contained high titers of the anti-Gal antibody indicated by the agglutination of glutaraldehyde-stabilized rabbit erythrocytes. The average titer of all SE-nonexposed hens was 9.0 corresponding to 1:5,120 when corrected for the initial dilution and expressed in conventional terms. Salmonella enteritidis exposure was associated with higher anti-Gal titers. The average anti-Gal titer for all SE-exposed hens was 10.0, corresponding to 1:10,240 in conventional terms; this difference was significant (P < or = 0.016). Diet efficiency type-associated differences in anti-Gal titers were not significant. Collectively, our data indicate that diet efficiency status is not associated with compromised Salmonella-specific immune responses. Rather it appears that the immune responses of diet efficient hens (R-) are also more efficient. This is because R- hens produced higher levels of O- and H-type antibody only as the result of direct exposure to living SE. On the contrary, R+ hens produced H-type antibody because of challenge with heat-killed SE, a circumstance that will not result in disease. Moreover, the hen type difference does not seem to occur at the expense of innate immunity as measured by anti-Gal antibody levels.