Short-term function and immune-protection of microencapsulated adult porcine islets with alginate incorporating CXCL12 in healthy and diabetic non-human primates without systemic immune suppression: A pilot study.

Replacement of insulin-producing pancreatic beta-cells by islet transplantation offers a functional cure for type-1 diabetes (T1D). We recently demonstrated that a clinical grade alginate micro-encapsulant incorporating the immune-repellent chemokine and pro-survival factor CXCL12 could protect and sustain the integrity and function of autologous islets in healthy non-human primates (NHPs) without systemic immune suppression. In this pilot study, we examined the impact of the CXCL12 micro encapsulant on the function and inflammatory and immune responses of xenogeneic islets transplanted into the omental tissue bilayer sac (OB; n = 4) and diabetic (n = 1) NHPs. Changes in the expression of cytokines after implantation were limited to 2-6-fold changes in blood, most of which did not persist over the first 4 weeks after implantation. Flow cytometry of PBMCs following transplantation showed minimal changes in IFNγ or TNFα expression on xenoantigen-specific CD4+  or CD8+  T cells compared to unstimulated cells, and these occurred mainly in the first 4 weeks. Microbeads were readily retrievable for assessment at day 90 and day 180 and at retrieval were without microscopic signs of degradation or foreign body responses (FBR). In vitro and immunohistochemistry studies of explanted microbeads indicated the presence of functional xenogeneic islets at day 30 post transplantation in all biopsied NHPs. These results from a small pilot study revealed that CXCL12-microencapsulated xenogeneic islets abrogate inflammatory and adaptive immune responses to the xenograft. This work paves the way toward future larger scale studies of the transplantation of alginate microbeads with CXCL12 and porcine or human stem cell-derived beta cells or allogeneic islets into diabetic NHPs without systemic immunosuppression.

Preliminary Studies of the Impact of CXCL12 on the Foreign Body Reaction to Pancreatic Islets Microencapsulated in Alginate in Nonhuman Primates.

BACKGROUND: We previously demonstrated that the incorporation of the chemokine CXCL12 into alginate microbeads supported long-term survival of microencapsulated auto-, allo-, and xenogeneic islets in murine models of diabetes without systemic immune suppression. The purpose of this study was to test whether CXCL12 could abrogate foreign body responses (FBRs) against alginate microbeads which were empty or contained autologous islets in healthy nonhuman primates (NHPs; n = 4). METHODS: Two NHPs received intraperitoneal implants of 400 000 alginate microbeads with or without CXCL12, and postimplantation immunological and histopathological changes were evaluated up to 6 months postimplantation. A similar evaluation of autologous islets in CXCL12-containing alginate microbeads was performed in NHPs (n = 2). RESULTS: CXCL12-containing alginate microbeads were associated with a markedly reduced FBR to microbeads. Host responses to microbead implants were minimal, as assessed by clinical observations, blood counts, and chemistry. Evaluation of encapsulated islets was limited by the development of necrotizing pancreatitis after hemipancreatectomy in 1 NHP. A limited number of functioning islets were detectable at 6 months posttransplantation in the second NHP. In general, empty microbeads or islet-containing beads were found to be evenly distributed through the intraperitoneal cavity and did not accumulate in the Pouch of Douglas. CONCLUSIONS: Inclusion of CXCL12 in alginate microbeads minimized localized FBR. The NHP autologous islet implant model had limited utility for excluding inflammatory/immune responses to implanted islets because of the complexity of pancreatic surgery (hemipancreatectomy) before transplantation and the need to microencapsulate and transplant encapsulated autologous islets immediately after pancreatectomy and islet isolation.

Alginate-microencapsulation of human stem cell-derived ß cells with CXCL12 prolongs their survival and function in immunocompetent mice without systemic immunosuppression.

Pancreatic ß-cell replacement by islet transplantation for the treatment of type 1 diabetes (T1D) is currently limited by donor tissue scarcity and the requirement for lifelong immunosuppression. The advent of in vitro differentiation protocols for generating functional ß-like cells from human pluripotent stem cells, also referred to as SC-ß cells, could eliminate these obstacles. To avoid the need for immunosuppression, alginate-microencapsulation is widely investigated as a safe path to ß-cell replacement. Nonetheless, inflammatory foreign body responses leading to pericapsular fibrotic overgrowth often causes microencapsulated islet-cell death and graft failure. Here we used a novel approach to evade the pericapsular fibrotic response to alginate-microencapsulated SC-ß cells; an immunomodulatory chemokine, CXCL12, was incorporated into clinical grade sodium alginate to microencapsulate SC-ß cells. CXCL12 enhanced glucose-stimulated insulin secretion activity of SC-ß cells and induced expression of genes associated with ß-cell function in vitro. SC-ß cells co-encapsulated with CXCL12 showed enhanced insulin secretion in diabetic mice and accelerated the normalization of hyperglycemia. Additionally, SC-ß cells co-encapsulated with CXCL12 evaded the pericapsular fibrotic response, resulting in long-term functional competence and glycemic correction (>150 days) without systemic immunosuppression in immunocompetent C57BL/6 mice. These findings lay the groundwork for further preclinical translation of this approach into large animal models of T1D.

SIVsm Tat, Rev, and Nef1: functional characteristics of r-GV internalization on isotypes, cytokines, and intracellular degradation.

BACKGROUND: Recombinant gas vesicles (r-GV) from Halobacterium sp. strain SD109 expressing cassettes with different SIVsm inserts, have potential utility as an effective antigen display system for immunogen testing in vivo and for initial epitope assessments in vitro. Previous mouse model studies demonstrated immunization with r-GV expressing selected exogenous sequences elicited a prolonged immune response. Here we tested segments from three SIVsm genes (tat, rev, and nef) each surface displayed by r-GV. As with HIV, for SIVsm the proteins encoded by tat, rev and nef respectively serve critical and diverse functions: effects on efficient viral RNA polymerase II transcription, regulation of viral gene expression and effects on specific signaling functions through the assembly of multiprotein complexes. Humoral responses to r-GVTat, Rev or Nef1 elicited in vivo, associated changes in selected cell cytokine production following r-GV internalization, and the capacity of J774A.1 macrophage cells to degrade these internalized display/delivery particles in vitro were examined. RESULTS: The in vivo studies involving r-GV immunizations and in vitro studies of r-GV uptake by J774A.1 macrophages demonstrated: (i) tests for antibody isotypes in immunized mice sera showed activation and re-stimulation of memory B cells, (ii) during long term immune response to the epitopes, primarily the IgG1 isotype was produced, (iii) in vitro, macrophage degradation of r-GV containing different SIVsm inserts occurred over a period of days resulting in an inherent slow breakdown and degradation of the SIVsm peptide inserts, (iv) vesicle specific GvpC, a larger protein, degraded more slowly than the recombinant peptide inserts and (v) in vitro uptake and degradation of the r-GV populations tested was associated with SIVsm insert specific patterns for cytokines IL-10, IL-12 and IL-18. CONCLUSIONS: Together these findings provide new information underscoring r-GV potential. They can clearly: display various exogenous peptides, be intracellularly degraded in vitro over a period of days, affect cell cytokine levels, and retain their self-adjuvanting capacity irrespective of the specific peptide expressed within the GvpC protein. These features support the cost effective generation of vaccine components, and provide a simple, self-adjuvanting system for assessing immune visibility of and specific responses to individual pathogen peptides.

Recombinant gas vesicles from Halobacterium sp. displaying SIV peptides demonstrate biotechnology potential as a pathogen peptide delivery vehicle.

BACKGROUND: Previous studies indicated that recombinant gas vesicles (r-GV) from a mutant strain of Halobacterium sp. NRC-1 could express a cassette containing test sequences of SIVmac gag derived DNA, and function as an antigen display/delivery system. Tests using mice indicated that the humoral immune response to the gag encoded sequences evoked immunologic memory in the absence of an exogenous adjuvant. RESULTS: The goal of this research was to extend this demonstration to diverse gene sequences by testing recombinant gas vesicles displaying peptides encoded by different SIV genes (SIVtat, rev or nef). Verification that different peptides can be successfully incorporated into the GvpC surface protein of gas vesicle would support a more general biotechnology application of this potential display/delivery system. Selected SIVsm-GvpC fusion peptides were generated by creating and expressing fusion genes, then assessing the resulting recombinant gas vesicles for SIV peptide specific antigenic and immunogenic capabilities. Results from these analyses support three conclusions: (i) Different recombinant gvpC-SIV genes will support the biosynthesis of chimeric, GvpC fusion proteins which are incorporated into the gas vesicles and generate functional organelles. (ii) Monkey antibody elicited by in vivo infection with SHIV recognizes these expressed SIV sequences in the fusion proteins encoded by the gvpC-SIV fusion genes as SIV peptides. (iii) Test of antiserum elicited by immunizing mice with recombinant gas vesicles demonstrated notable and long term antibody titers. The observed level of humoral responses, and the maintenance of elevated responses to, Tat, Rev and Nef1 encoded peptides carried by the respective r-GV, are consistent with the suggestion that in vivo there may be a natural and slow release of epitope over time. CONCLUSION: The findings therefore suggest that in addition to providing information about these specific inserts, r-GV displaying peptide inserts from other relevant pathogens could have significant biotechnological potential for display and delivery, or serve as a cost effective initial screen of pathogen derived peptides naturally expressed during infections in vivo.

Cassette-based presentation of SIV epitopes with recombinant gas vesicles from halophilic archaea.

In earlier studies we demonstrated recombinant gas vesicles from Halobacterium sp. NRC-1, expressing a model six amino acid insert, or native vesicles displaying chemically coupled TNP, each were immunogenic, and antigenic. Long-lived responses displaying immunologic memory were elicited without exogenous adjuvant. Here we report the generation and expression of cassettes containing SIV derived DNA. The results indicate a cassette-based display/delivery system derived from recombinant halobacterial gas vesicle genes is highly feasible. Data specifically support four conclusions: (i) Recombinants carrying up to 705 bp of SIV DNA inserted into the gvpC gene form functional gas vesicles; (ii) SIV peptides contained as part of the expressed recombinant, surface exposed GvpC protein are recognized by antibody elicited in monkeys exposed to native SIV in vivo; (iii) in the absence of adjuvant, mice immunized with the recombinant gas vesicle (r-GV) preparations mount a solid, titratable antibody response to the test SIV insert that is long lived and exhibits immunologic memory; (iv) recombinant organelles, created through the generation of cassettes encoding epitopes inserted into the gvpC DNA, can be used to construct a multiepitope display (MED) library, a potentially cost effective vehicle to express and deliver peptides of SIV, HIV or other pathogens.