ALBERT

Description: A serious complication in the treatment X-linked bleeding disorder hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), which compromise traditional replacement therapy. We previously developed an Oral immunotherapy (OTI) based on repeated uptake of a mixture of lettuce plant cells transgenic for heavy chain (HC) or C2 domain of human FVIII fused to cholera toxin B (CTB) subunit [Blood 124:1659; Plant Biotechnol J. 16:1148]. Fusion proteins were transgenically expressed in the chloroplasts. Repeated oral uptake of a mixture of freeze-dried powder of lettuce cells accomplished antigen delivery to the immune system of the small intestine by targeting of the GM1 receptor that is highly expressed on the surface of the gut epithelium, resulting in induction of regulatory T cells (Treg) that suppress inhibitor formation upon subsequent intravenous (iv) FVIII replacement therapy. An alternative to oral antigen delivery is the oral delivery of immune modulatory antibodies. Here, we compared the plant cell-based method with oral delivery of anti-CD3, which has been successful in murine models of autoimmune disease and is currently evaluated in clinical trials. Unlike in iv administration, oral anti-CD3 does not systemically deplete T cells. Hemophilia A BALB/c mice (F8 e16 gene deletion) received oral gavage of a mixture of CTB-FVIII-HC/-C2 (1.5 µg/antigen) expressing lettuce leaf cells 2x/week for 9 weeks. Starting at 4 weeks into the experiment, 1 IU/mouse of BDD-FVIII (Xyntha) was given iv, once per week for 5 weeks. Alternatively, following a published protocol that was successful in other models, 5 µg of hamster anti-murine CD3 was given by oral gavage daily for 5 straight days, followed by 5 weekly iv injections of BDD-FVIII. Control animals (no OTI) developed inhibitors with an average titer of 18 ± 3 BU/ml (n=16). Of these, 88% were high-titer (i.e 〉5 BU/ml, up to 43 BU/ml). Inhibitor formation was significantly reduced in plant cell-treated mice (10 ± 2.5 BU/ml, n=17), with 47% showing no or low-titer inhibitors (

Description: Adeno-associated viral (AAV) vectors are currently evaluated in multiple Phase III clinical trial for the treatment of hemophilia and neuromuscular disorders. A major concern is the potential for immune responses. Viral vectors are initially sensed by the innate immune system, which shapes subsequent adaptive immune responses. Particularly, toll-like receptors (TLRs) have been reported as major sensors of pathogens during innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMPs). Our previous studies found that cross-priming of AAV capsid-specific CD8+ T cells depended on TLR9-MyD88 pathway. TLR9 is an endosomal DNA receptor that responds most potently to unmethylated CpG motifs as found in bacterial and viral DNA. Similarly, others documented TLR9-dependent CD8+ T cell responses against non-secreted transgene products such as LacZ and hemagglutinin upon muscle-directed AAV gene transfer. Similarly, we published that CD8+ T cell responses to a secreted ovalbumin (ova) transgene product were substantially reduced (although not entirely eliminated) upon muscle gene transfer in TLR9-deficient mice [J Innate Immun. 7:302-14]. For those studies, we had used a self-complementary scAAV genomes, which we found to more strongly activate TLR9 than conventional single-stranded ssAAV vectors. Here, we performed intramuscular injections of 3 doses of ssAAV1-CMV-ova vector (2X1010, 2X1011 and1X1012 vg) in wild-type (WT), TLR9-/-, or MYD88-/- C57BL/6 mice. Using MHC tetramer (H2-Kb -SIINFEKL), ova-specific CD8+ T cell frequencies were monitored in peripheral blood for up to 6 weeks. As expected from prior studies, TLR9-/- mice showed a substantially reduced response (1.2% tetramer+ of CD8) at the low dose when compared to WT (12% tetramer+ of CD8) animals (p

Description: Inhibitor formation is the most serious complication of FVIII replacement therapy for hemophilia A. The long-standing "danger theory" posits that inhibitors may form as part of collateral damage from immune response to a primary challenge such as an infection or vaccination. Innate immune signaling, for example through Toll-like receptors (TLRs), could be one way of triggering or reinforcing unwanted immune responses. However, the danger hypothesis has been contradicted by recent reports showing no increase in inhibitor formation in boys and animal models with hemophilia when FVIII was administered concurrently with vaccines. The aim of this study was to elucidate the influence of TLR9 signaling on FVIII inhibitor formation in hemophilia A mice. Hemophilia A (F8e16-/-) B6/129 mice were co-injected IV with FVIII (1.5 IU) and ODN-1826 (a class B CpG oligodeoxynucleotide, 50 µg), which is a TLR9 agonist. Control mice were naïve or received FVIII only. Blood samples and spleens were collected for Bethesda assay and flow cytometry analysis 3h, 24h, 2, 3 and 7 days or 2, 4, 6 and 8 weeks after a single or repeated once-weekly injections. After four weeks, mice co-injected with FVIII and ODN-1826 (n=4) showed ~15-fold higher inhibitor titers (median 2667 BU/mL) than mice injected with FVIII only (median 181.4 BU/mL; n=15). We also found significantly higher T follicular helper (Tfh; CD4+CXCR5+PD1+Bcl6―) [F (7, 96) = 9.801, p