Joanita Figueredo

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8+ T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.

Impact of Preexisting Vector Immunity on the Efficacy of Adeno-Associated Virus-Based HIV-1 Gag Vaccines

Vectors based on primate-derived adeno-associated virus (AAV) are being considered in the development of genetic vaccines against a number of diseases including infection with HIV-1. Preexisting immunity to the vaccine carrier as a result of natural infections could potentially compromise vaccine efficacy. This study evaluates the impact of neutralizing antibodies against AAV capsids on the ability of HIV-1 Gag-expressing vectors to elicit transgene-specific T and B cell responses. Mice were passively transferred with pooled human immunoglobulin at various doses to simulate human antivector humoral immunity. Vectors based on serotype 2, which were evaluated in the clinic, were compared with those created from the novel monkey isolates AAV7 and AAV8. Inhibition of AAV2-directed Gag responses occurred at doses of human immunoglobulin 10- to 20-fold less than was required to inhibit immunogenicity of AAV7 and AAV8 vectors. Cynomolgus macaques were screened for preexisting immunity to AAV7 and AAV8 and sera from individual animals were passively transferred into mice that were analyzed for AAV vaccine efficacy. There was a correlation between the level of preexisting capsid neutralizing titers and diminution of vaccine efficacy; sera from a number of animals with no detectable neutralizing antibodies showed partial vaccine inhibition, suggesting that the in vitro assay is less sensitive than the in vivo passive transfer assay for detecting neutralizing antibodies to AAV.

Adenovirus-based vaccine prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates robust immune responses in macaques

Abstract A ferret model of severe acute respiratory syndrome (SARS)-CoV infection was used to evaluate the efficacy of an adenovirus vaccine. Animals were subjected to heterologous prime-boost using vectors from human serotype 5 and chimpanzee derived adenoviruses (human AdHu5 and chimpanzee AdC7) expressing spike protein followed by intranasal challenge with SARS-CoV. Vaccination led to a substantial reduction in viral load and prevented the severe pneumonia seen in unvaccinated animals. The same prime-boost strategy was effective in rhesus macaques in eliciting SARS-CoV specific immune responses. These data indicate that a heterologous adenovirus-based prime-boost vaccine strategy could safely stimulate strong immunity that may be needed for complete protection against SARS-CoV infection.

Corrigendum to “Biology of AAV Serotype Vectors in Liver-Directed Gene Transfer to Nonhuman Primates”

The authors regret the following errors: On page 80, the second sentence in the legend to Fig. 2 should read bAST (the right y axes) and rhCG (the left y axes) levels at different time points after vector infusion from each of eight study subjects are presentedQ instead of bAST (the left y axes) and rhCG (the right y axes) levels at different time points after vector infusion from each of eight study subjects are presented.Q On page 80, the right y axes label in Fig. 2 should read bAST (IU/L)Q instead of bAST (IU/ml).Q

112. Strong Transgene-Specific Immune Responses Can Be Elicited with Novel Adeno-Associated Virus Vectors in Mice

Vectors based on recombinant adeno-associated virus (rAAV) have been widely appreciated for gene therapy application, in which the sustained expression of self-genes has been successfully achieved in a variety of animal models. However, the potential of rAAV vectors as vaccine carriers has only recently been recognized. We have isolated several novel AAV serotypes from primates i.e AAVs 7, 8, and 9. Therefore, it is of our interest to develop potential vaccines for human immunodeficiency virus type 1 (HIV-1) based on rAAV vectors of these new serotypes. We have constructed a panel of rAAV vectors based on different AAV serotypes (including AAV2/1, AAV2, AAV2/5, AAV2/7, AAV2/8, and AAV2/9), which all encoded HIV-1 gag p17 and p24 (designated as gagshort). BALB/c mice were i.m. immunized with this panel of AAVHIVgagshort vectors at the dose of E11 genome copies/mouse. 2, 3, and 4 weeks after immunization, splenocytes were harvested and stimulated with the H-2d restricted gag-specific CD8 T-cell epitope in intracellular IFN-g staining. Overall, AAV2/7 and AAV2/8 vaccine vectors elicited the highest levels of gag-specific CD8 T-cell responses; while AAV2/1 and AAV2/9 vaccine vectors were able to induce intermediate levels of gag-specific CD8 T-cell responses. Gag-specific CD8 T-cell responses induced with AAV2/5 and AAV2/2 vaccine vectors were barely detectable. In addition, serum samples from immunized mice were collected and total IgG responses to gag p24 were measured by ELISA. Overall, AAV2/7 and AAV2/9 vaccine vectors yielded the strongest IgG responses against p24; while AAV2/8, AAV2/1, and AAV2/5 vaccine vectors produced the intermediate IgG responses against p24. Surprisingly, there was no detectable p24-specific IgG response in serum samples from mice immunized with AAV2/2HIVgagshort vector, even though this vector yielded very high level of p24 expression in transduced cells in vitro. Interestingly, different patterns of p24-specific IgG1 vs IgG2a responses were observed in serum samples from mice immunized with HIVgagshort vaccine vectors based on different AAV serotypes. Furthermore, significant inflammation and infiltration in the mouse muscles injected with AAV2/7 and AAV2/8 vaccine vectors were observed at 4 weeks after i.m. injection by H/E staining. Taken together, these data suggested that strong transgene-specific immune responses can be elicited with novel rAAV vectors in mice. The immunogenicity of these vectors based on novel AAV serotypes will be examined in nonhuman primates.

1092. Adenovirus-Based Vaccine Prevents Pneumonia in Ferrets Challenged with the SARS Coronavirus

Top of pageAbstract The human severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiologic agent responsible for the spread of the acute respiratory syndrome in Asia and Canada that claimed several human lives in 2003. A ferret model of SARS-CoV infection was used to evaluate the efficacy of an adenovirus-based vaccine. Ferrets infected with a clinical isolate of SARS-CoV via intranasal inhalation developed a syndrome similar to that observed in humans that consisted of fever and clinical signs of toxicity. Virus replicated in lung and was recovered in nasal washes. Patchy but widely disseminated bronchopneumonia developed characterized by infiltration of neutrophils and monocytes. Animals were vaccinated with recombinant adenoviruses expressing the Spike protein of SARS-CoV prior to challenge with SARS-CoV. The most impressive therapeutic results were obtained when the animals were primed with a human adenovirus expressing Spike and boosted with a serologically distinct adenovirus from chimpanzee also expressing the Spike antigen. Following challenge with SARS-CoV the acute fever was lessened and clinical sequalae were diminished. Viral loads in lung and nasal washes were down 6 logs as compared to animals vaccinated with a control vector. There was no evidence of gross lung pathology at necropsy and histological findings were limited to mild focal inflammation in alveolar septa and around some small airways. The same prime boost strategy was also effective in rhesus macaques and resulted in SARS-CoV neutralizing antibody titers of up to 1/2560 and |[gamma]|-INF positive T cells reaching 12,000 SFC per million PBMCs as assessed by ELISPOT. These data indicate that a heterologous adenovirus-based prime boost vaccine strategy could stimulate a strong immunity that may be needed for complete protection against SARS-CoV infection.

146. T Cell Response Against the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

Top of pageAbstract Gene therapy for cystic fibrosis airway disease appears to be the most promising curative option and much effort has been focused on developing gene transfer vectors combined with ways to manipulate the barriers that the airway poses to gene transfer. However, there has been no evaluation of the possibility of a T cell response against the CFTR gene in the context of gene therapy. Although this may not be a limitation in the majority of CF patients which express residual amounts of CFTR, the likelihood of immune responses against CFTR must be investigated. As such we designed a peptide library of the human CFTR consisting of 294 peptides of 15 amino acids (aa) each with an overlapping 10 aa sequence. In the first instance we evaluated the immune response generated by an adenovirus (Ad) vector expressing the hCFTR gene under the transcriptional control of the chicken b-actin promoter in CFTR knockout as well as CFTR heterozygote mice. Specifically, 5e10 particles of an Ad vector were injected intramuscularly into the right hindlimb of CFTR knockout and CFTR heterozygote mice. Eight days later the mice were sacrificed and the spleens harvested. T cell response was assayed using IFN-g ELISPOT following stimulation of splenocytes with 15 pools (20 peptides/pool, final concentration of 2.5 mg/ml) of the CFTR peptide library. A T cell response was observed in 7 out of 15 peptide pools and we are currently screening those in an attempt to map the epitopes. The pattern of the T cell immune response observed was almost similar in both groups. However, we identified a minor epitope in the CFTR knockout group that did not show any activity in the CFTR heterozygote group suggesting that high levels of CFTR may not compromise pre-existing tolerance. We are examining whether the T cell response is CD4 or CD8 cell mediated and also evaluating T cell activation against CFTR following an intranasal instillation of varying doses of Ad-CFTR vector. In conclusion, this study will help elucidate whether a T cell response is generated following CFTR gene transfer and assess how this will affect the outcome of CFTR gene therapy in a clinical setting.