Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (~1mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ~2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP+ regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ~870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft2 per annum yielding 24,000–36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.
• Factor VIII antigens can be expressed in chloroplasts and bioencapsulated in plant cells.• Oral delivery of plant cells expressing FVIII domains suppresses and reverses inhibitor formation in mice with hemophilia A by induction of CD4 1 regulatory T cells.Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 mg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor b and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4 1 CD25 1 and CD4 1 CD25 2 T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII. (Blood. 2014;124(10):1659-1668
• Coadministering FIX orally and systemically induces tolerance via complex immune regulation, involving tolerogenic dendritic and T-cell subsets.• Induced CD4 Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed.
Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.
Renewed interest in chlamydia vaccination has revealed the need for a greater understanding of the seroprevalence of chlamydial infection in US populations. We used a Chlamydia trachomatis elementary body (EB)-based enzyme-linked immunosorbent assay to define the characteristics of the humoral immune response and to determine seroprevalence. Two groups were analyzed: one consisting of patients with current, laboratory confirmed, genital chlamydial infection (n = 98) and one group of individuals whose chlamydia infection history was unknown (n = 367). C. trachomatis seropositivity was detected in 90% of the infected group and in 31% of the chlamydia-unknown group. IgG1 and IgG3 comprised the predominant anti-Chlamydia serum antibody responses. Serum IgA1 responses were variably positive, and individuals were rarely positive for anti-chlamydia IgG2, IgG4 or IgA2. The magnitude of the IgG1 and IgG3 responses was greatest in female and African American individuals and was sustained for at least 6 months. Antibody responses were not serovar restricted or confounded by Chlamydia pneumoniae cross-reactivity.
Summary Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation.
SummaryThe impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300‐fold higher in C and CN lines than in N, but all accumulated ~150‐fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65–120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32‐fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000‐fold in CN; 7784 transcripts by up to 1400‐fold in N; and 5224 transcripts by as much as 2200‐fold in C. Transporter‐related transcripts were induced, and cell cycle‐associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT‐PCR. The mechanism underlying these large changes likely involves metabolite‐mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.
The rhesus macaque ( Macaca mulatta) has become a popular animal model for several human infectious diseases, such as HIV (modeled by SIV infection), hepatitis, and malaria. Investigation of T-cell responses in experimental infectious diseases in rhesus macaques has benefited from an expanding understanding of the diversity of macaque MHC class I heavy chains and the restriction of antigen presentation by macaque class I molecules. Here we add to this understanding with the first nucleotide sequences of M. mulatta beta(2)-microglobulin (beta(2)m) mRNA, including a portion of the 3'-untranslated region (3'UTR). In pairwise comparison, the beta(2)m protein of M. mulatta differs from human and chimpanzee beta(2)m by nine amino-acid substitutions (92% identity), and from Macaca fascicularis by one amino-acid difference in the signal peptide region (99% identity). Allelic variations were identified at one site in the 3'UTR. A structural analysis of human or chimpanzee beta(2)m and M. mulatta beta(2)m suggests that the differences cluster in three solvent-exposed clusters and do not involve contacts with the class I heavy chain. We predict that human and macaque beta(2)m should bind interchangeably with the class I heavy chains of the other species, and show that four M. mulatta class I alleles form cell surface complexes with human beta(2)m. Further, we predict that W6/32 (a monoclonal antibody that recognizes a combined epitope of some class I heavy chains and beta(2)m with a subtle species dependence) should bind similarly human or macaque class I molecules that are bound with beta(2)m of either species, supported by evidence of recognition of both heterologous and homologous complexes of macaque class I heavy chains. Our findings contribute to the growing understanding of rhesus macaque histocompatibility antigens and antigen presentation, and to the phylogeny of beta(2)m in primates.
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