A CTB-SARS-CoV-2-ACE-2 RBD Mucosal Vaccine Protects Against Coronavirus Infection.

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.

Peritoneal carcinomatosis in gastric cancer: Are Hispanics at higher risk?

BACKGROUND: A recent study from our group identified Hispanic race/ethnicity as an independent predictor of peritoneal carcinomatosis (PC) in gastric cancer. We sought to identify the tumor factors that might contribute to this strong association in Hispanics. METHODS: California Cancer Registry data were used to identify patients diagnosed with gastric adenocarcinoma from 2004 to 2014. Logistic regression analyses were performed to determine odds ratios for cancer stage, tumor location, grade, histology, and PC. RESULTS: Of 16,275 patients with gastric adenocarcinoma who met inclusion criteria, 6463 (39.7%) were non-Hispanic White (NHW), 4953 (30.4%) were Hispanic, 1020 (6.3%) were non-Hispanic Black (NHB), and 3915 (23.6%) were Asian/other. Compared to NHW, Hispanics were more likely to have a poorly differentiated grade (65.9% vs. 57.6%; p < .001), signet ring adenocarcinoma (28.1% vs. 17.6%; p < .001) and stage IV (51.9% vs. 45.0%; p < .001) gastric cancer. The proportion of stage IV patients with PC was also significantly higher in Hispanics compared to NHW, NHB, and Asian/other (28.5% vs. 16.6%, 20.5%, and 25.2%, respectively; p < .001). CONCLUSIONS: Hispanic ethnicity is an independent predictor of aggressive tumor phenotype and PC. Disproportionate incidence of signet ring adenocarcinoma and PC highlight the need to explore the genomic differences in Hispanic gastric cancer.

En Balance: The Contribution of Physical Activity to the Efficacy of Spanish Diabetes Education of Hispanic Americans with Type 2 Diabetes.

PURPOSE: En Balance, a culturally sensitive diabetes education program, improves glycemic control in Hispanics with type 2 diabetes. The program emphasized diet, physical activity, and other factors important for glycemic control. However, the individual contributions of these education factors are unclear. The purpose of this study is to assess the contribution of physical activity to the success of En Balance in improving the health of Mexican Americans with type 2 diabetes. METHODS: A retrospective study was conducted with plasma samples collected pre- and post-3-month study. Samples from 58 (18 males and 40 females) Hispanic subjects with type 2 diabetes were analyzed for the concentration of kynurenines, known to decrease in response to exercise. After three months, health outcomes for the active group (decreased kynurenines) and the rest of the cohort were evaluated by paired Wilcoxon signed-rank test. RESULTS: Half of the subjects had increased kynurenine levels at the end of the educational program. We found that the subjects in the active group with decreased kynurenine concentrations displayed statistically greater improvements in fasting blood glucose, A1C, cholesterol, and triglycerides despite weight loss being higher in the group with increased kynurenine concentrations. CONCLUSIONS: En Balance participants with decreased kynurenine levels had significantly improved glycemic control. These data suggest that physical activity significantly contributes to the success of the En Balance education program. This analysis indicates that diabetes public health educators should emphasize the benefit of physical activity on glycemic control even in the absence of major weight loss.

Extracellular vimentin modulates human dendritic cell activation.

Vimentin is an intermediate filament protein traditionally considered to be an intracellular protein with a structural role. However, recent evidence suggests that vimentin can also be found outside the cell in disease conditions such as cancer, traumatic tissue injury, and inflammation. Extracellular vimentin was previously found to stimulate innate immunity by increasing monocyte and macrophage ability to kill bacteria. However, vimentin has also been previously found to decrease neutrophil infiltration into inflamed tissue. How extracellular vimentin affects the initiation of adaptive immune responses is unknown. Initiation of adaptive immunity involves priming of naïve T cells by antigen-presenting cells, the most effective of which are dendritic cells (DCs). In this study, we demonstrate how extracellular vimentin modulates lipopolysaccharide (LPS) - induced activation of human DCs. Using cytometric bead arrays, we show that extracellular vimentin decreases LPS-activated DC secretion of pro-inflammatory cytokines IL-6 and IL-12 while increasing secretion of the anti-inflammatory cytokine IL-10. Using flow cytometry, we show that extracellular vimentin does not significantly affect LPS-induced DC surface expression of MHC I (HLA-ABC) or MHC II (HLA-DR) presentation molecules, costimulatory factors (CD80, CD86), or the DC maturation marker (CD83). Further, LPS-stimulated DCs co-cultured with allogeneic naïve CD4 + T cells (Th0) induced less secretion of the pro-inflammatory Th1 effector cytokine IFN-γ in the presence of vimentin than in the presence of LPS alone. This result suggests that vimentin reduces Th1 differentiation. Taken together, our data suggest that extracellular vimentin may inhibit pro-inflammatory adaptive immune responses, by blocking DC secretion of pro-inflammatory cytokines. Thus, extracellular vimentin may play an important role in cancer or trauma-complications by inducing suppression of the adaptive immune response. In a positive sense, the presence of extracellular vimentin may prevent tissue-damage from contributing to the development of autoimmunity. Consequently, extracellular vimentin may become a novel drug target for treatment of a variety of pro- and anti-inflammatory disease conditions.

Predicting methotrexate resistance in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an incurable, systemic autoimmune disease that decreases quality of life and can lead to severe disability. While there are many medications available to treat RA, the first-line of therapy is low-dose methotrexate (MTX), a small molecule disease-modifying anti-rheumatic drug (DMARD). MTX is the recommended therapy due to its affordability and efficacy in reducing symptoms in most RA patients. Unfortunately, there is great person-to-person variability in the physiological response to MTX, with up to 50% of patients showing little response to the medication. Thus, many RA patients initially placed on MTX do not experience an adequate reduction of symptoms, and could have benefited more in both the short and long terms if initially prescribed a different drug that was more effective for them. To combat this problem and better guide treatment decisions, many research groups have attempted to develop predictive tools for MTX response. Currently, there is no reliable, clinical-grade method to predict an individual's response to MTX treatment. In this review, we describe progress made in the area of MTX non-response/resistance in RA patients. We specifically focus on application of the following elements as predictive markers: proteins related to MTX transport and function, intracellular MTX concentration, immune cell frequencies, cytokines, and clinical factors.

Mechanism of chimeric vaccine stimulation of indoleamine 2,3-dioxygenase biosynthesis in human dendritic cells is independent of TGF-ß signaling.

Cholera toxin B subunit fusion to autoantigens such as proinsulin (CTB-INS) down regulate dendritic cell (DC) activation and stimulate synthesis of DC immunosuppressive cytokines. Recent studies of CTB-INS induction of immune tolerance in human DCs indicate that increased biosynthesis of indoleamine 2,3-dioxygenase (IDO1) may play an important role in CTB-INS vaccine suppression of DC activation. Studies in murine models suggest a role for transforming growth factor beta (TGF-ß) in the stimulation of IDO1 biosynthesis, for the induction of tolerance in DCs. Here, we investigated the contribution of TGF-ß superfamily proteins to CTB-INS induction of IDO1 biosynthesis in human monocyte-derived DCs (moDCs). We show that CTB-INS upregulates the level of TGF-ß1, activin-A and the TGF-ß activator, integrin αvß8 in human DCs. However, inhibition of endogenous TGF-ß, activin-A or addition of biologically active TGF-ß1, and activin-A, did not inhibit or stimulate IDO1 biosynthesis in human DCs treated with CTB-INS. While inhibition with the kinase inhibitor, RepSox, blocked SMAD2/3 phosphorylation and diminished IDO1 biosynthesis in a concentration dependent manner. Specific blocking of the TGF-ß type 1 kinase receptor with SB-431542 did not arrest IDO1 biosynthesis, suggesting the involvement of a different kinase pathway other than TGF-ß type 1 receptor kinase in CTB-INS induction of IDO1 in human moDCs. Together, our experimental findings identify additional immunoregulatory proteins induced by the CTB-INS fusion protein, suggesting CTB-INS may utilize multiple mechanisms in the induction of tolerance in human moDCs.

The role of TGF-beta signaling in dendritic cell tolerance.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine present in vertebrate and invertebrate organisms that functions in numerous physiological and pathological processes. TGF-ß impacts all the cells of the immune system, and of the three known TGF-ß isoforms, TGF-ß1 is the predominant isoform expressed in immune cells. TGF-ß1 is known to play a pivotal role in the function of all immune cells especially in the regulation of T cell development and in the induction of immunological tolerance in dendritic cells (DCs). Based on the importance of DCs in regulation of the innate and adaptive arms of the immune system, in this review we explore the regulatory functions of TGF-ß required for establishment and maintenance of DC-mediated immune tolerance.

Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1ß.

Palmitic acid (PA) and other saturated fatty acids are known to stimulate pro-inflammatory responses in human immune cells via Toll-like receptor 4 (TLR4). However, the molecular mechanism responsible for fatty acid stimulation of TLR4 remains unknown. Here, we demonstrate that PA functions as a ligand for TLR4 on human monocyte derived dendritic cells (MoDCs). Hydrophobicity protein modeling indicated PA can associate with the hydrophobic binding pocket of TLR4 adaptor protein MD-2. Isothermal titration calorimetry quantified heat absorption that occurred during PA titration into TLR4/MD2, indicating that PA binds to TLR4/MD2. Treatment of human MoDCs with PA resulted in endocytosis of TLR4, further supporting the function of PA as a TLR4 agonist. In addition, PA stimulated DC maturation and activation based on the upregulation of DC costimulatory factors CD86 and CD83. Further experiments showed that PA induced TLR4 dependent secretion of the pro-inflammatory cytokine IL-1ß. Lastly, our experimental data show that PA stimulation of NF-κB canonical pathway activation is regulated by TLR4 signaling and that reactive oxygen species may be important in upregulating this pro-inflammatory response. Our experiments demonstrate for the first time that PA activation of TLR4 occurs in response to direct molecular interactions between PA and MD-2. In summary, our findings suggest a likely molecular mechanism for PA induction of pro-inflammatory immune responses in human dendritic cells expressing TLR4.

The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus.

Dendritic cells (DCs) are the dominant class of antigen-presenting cells in humans and are largely responsible for the initiation and guidance of innate and adaptive immune responses involved in maintenance of immunological homeostasis. Immature dendritic cells (iDCs) phagocytize pathogens and toxic proteins and in endosomal vesicles degrade them into small fragments for presentation on major histocompatibility complex (MHC) II receptor molecules to naïve cognate T cells (Th0). In addition to their role in stimulation of immunity, DCs are involved in the induction and maintenance of immune tolerance toward self-antigens. During activation, the iDCs become mature. Maturation begins when the DCs cease taking up antigens and begin to migrate from their location in peripheral tissues to adjacent lymph nodes or the spleen where during their continued maturation the DCs present stored antigens on surface MHCII receptor molecules to naive Th0 cells. During antigen presentation, the DCs upregulate the biosynthesis of costimulatory receptor molecules CD86, CD80, CD83, and CD40 on their plasma membrane. These activated DC receptor molecules bind cognate CD28 receptors presented on the Th0 cell membrane, which triggers DC secretion of IL-12 or IL-10 cytokines resulting in T cell differentiation into pro- or anti-inflammatory T cell subsets. Although basic concepts involved in the process of iDC activation and guidance of Th0 cell differentiation have been previously documented, they are poorly defined. In this review, we detail what is known about the process of DC maturation and its role in the induction of insulin-dependent diabetes mellitus autoimmunity.

The function of myeloid dendritic cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that causes joint pain, inflammation, and loss of function. Disease pathogenesis involves activation and proliferation of autoreactive pro-inflammatory effector T cells. While the details of RA onset and progression remain controversial, dendritic cell (DC) numbers dramatically increase in the synovial joint tissues of RA patients. Based on their key functions as antigen-presenting cells and inducers of T cell differentiation, DCs may play an important role in the initiation of joint inflammation. Myeloid DC contributions are likely central to the development of RA, as they are more efficient at antigen presentation in comparison with their closely related cousins, plasmacytoid DCs. Synovial fluid in the joints of RA patients is enriched with pro-inflammatory cytokines and chemokines, which may stimulate or result from DC activation. Epidemiological evidence indicates that smoking and periodontal infection are major environmental risk factors for RA development. In this review, factors in the synovial environment that contribute to altered myeloid DC functions in RA and the effects of environmental risk factors on myeloid DCs are described.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes.

High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1ß. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1ß, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1ß secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients.

The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity.

Indoleamine 2, 3-dioxygenase (IDO) is the first and rate limiting catabolic enzyme in the degradation pathway of the essential amino acid tryptophan. By cleaving the aromatic indole ring of tryptophan, IDO initiates the production of a variety of tryptophan degradation products called "kynurenines" that are known to exert important immuno-regulatory functions. Because tryptophan must be supplied in the diet, regulation of tryptophan catabolism may exert profound effects by activating or inhibiting metabolism and immune responses. Important for survival, the regulation of IDO biosynthesis and its activity in cells of the immune system can critically alter their responses to immunological insults, such as infection, autoimmunity and cancer. In this review, we assess how IDO-mediated catabolism of tryptophan can modulate the immune system to arrest inflammation, suppress immunity to cancer and inhibit allergy, autoimmunity and the rejection of transplanted tissues. Finally, we examine how vaccines may enhance immune suppression of autoimmunity through the upregulation of IDO biosynthesis in human dendritic cells.

Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic ß-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

Persistent suppression of type 1 diabetes by a multicomponent vaccine containing a cholera toxin B subunit-autoantigen fusion protein and complete Freund's adjuvant.

Data presented here demonstrate multifunctional vaccination strategies that harness vaccinia virus mediated delivery of a gene encoding an immunoenhanced diabetes autoantigen in combination with complete Freund's adjuvant (CFA) that can maintain safe and durable immunologic homeostasis in NOD mice. Systemic coinoculation of prediabetic mice with recombinant vaccinia virus rVV-CTB::GAD and undiluted or 10-fold diluted CFA demonstrated a significant decrease in hyperglycemia and pancreatic islet inflammation in comparison with control animals during 17-61 and 17-105 weeks of age, respectively. Synergy in these beneficial effects was observed during 43-61 and 61-105 wks of age, respectively. Inflammatory cytokine and chemokine levels in GAD-stimulated splenocytes isolated from vaccinated mice were generally lower than those detected in unvaccinated mice. The overall health and humoral immune responses of the vaccinated animals remained normal throughout the duration of the experiments.

Autoantigen based vaccines for type 1 diabetes.

Type 1 diabetes is an organ-specific autoimmune disease caused by chronic inflammation (insulitis), which damages the insulin producing ß-cells of the pancreatic Islets of Langerhans. Dendritic cells (DCs) are generally the first cells of the immune system to process ß-cell autoantigens and, by promoting autoreactivity, play a major role in the onset of insulitis. Although no cure for diabetes presently exists, the onset of insulitis can be diminished in the non-obese diabetic (NOD) mouse type 1 diabetes model by inoculation with endogenous ß-cell autoantigens. These include the single peptide vaccines insulin, GAD(65) (glutamic acid decarboxylase), and DiaPep277 (an immunogenic peptide from the 60-kDa heat shock protein). DiaPep277 is the only autoantigen so far to demonstrate positive results in human clinical trials. Diamyd (an alum adjuvant + recombinant GAD(65) protein formulation) has shown great promise for suppressing ß-cell autoreactivity in phase I and II clinical trials. While Diamyd preserved residual insulin secretion in early-onset type 1 diabetes patients, it did not reduce the amounts of insulin required to maintain euglycemia. Recently, multi-component vaccines composed of the anti-inflammatory cytokine (IL-10) and insulin or GAD(55) linked to an immunostimulatory molecule, the cholera toxin B subunit, were shown to safely and completely inhibit diabetes onset in NOD mice. This result suggests that multi-component vaccine strategies are promising for prevention and reversal of diabetes autoimmunity in humans. Here we focus on the development of autoantigen vaccines for type 1 diabetes and demonstrate that multi-component vaccines are promising candidates for type 1 diabetes clinical studies.

Autoantigens plus interleukin-10 suppress diabetes autoimmunity.

BACKGROUND: Recombinant vaccinia virus (rVV) strains expressing the immunomodulatory cholera toxin B subunit (CTB) fused to the autoantigen glutamic acid decarboxylase (GAD) or the immunosuppressive cytokine interleukin-10 (IL-10) were independently able to generate only low levels of immune suppression of type 1 diabetes mellitus (T1DM). Here we suggest that a vaccinia virus (VV)-mediated combination of CTB::GAD fusion and IL-10 proteins promises a effective and durable immunotherapeutic strategy for T1DM. METHODS: To explore this hypothesis, a CTB::GAD fusion gene was co-delivered with a gene encoding IL-10 by rVV infection (rVV-CTB::GAD + rVV-IL10) into 5-7-week-old non-obese diabetic (NOD) mice. The mice were assessed for vaccine protection against development of hyperglycemia from 12 to 64 weeks of age by assessment of pancreatic inflammation (insulitis) and splenocyte-secreted interferon-gamma and IL-10 cytokine levels. RESULTS: By 36 weeks of age, from 54% to 80% of the mice in the negative control animal groups (either mock-infected or inoculated with unrelated plasmid or VV) had developed hyperglycemia. Similarly, no statistically significant improvement in protection against diabetes onset was achieved by inoculation with VV expressing CTB::GAD or IL-10 independently. Surprisingly, only 20% of mice co-inoculated with rVV-CTB::GAD + rVV-IL10 developed hyperglycemia by 28 weeks of age. Other treatment groups developed hyperglycemia by 32-36 weeks. After 36 weeks, diabetes incidence no longer increased in any groups until the end of experiment at 64 weeks of age. Histological analysis of pancreatic tissues of hyperglycemic mice revealed high levels of intra-islet insulitis. Analysis of insulitis at termination of the experiment showed that euglycemic mice co-inoculated with VV expressing CTB::GAD and IL-10 had more effectively reduced inflammation in comparison with the other groups. CONCLUSIONS: A combinatorial vaccination strategy based on VV co-delivery of genes encoding the immunoenhanced autoantigen CTB::GAD and the anti-inflammatory cytokine IL-10 can maintain effective and durable euglycemia and immunological homeostasis in NOD mice with prediabetes.

Expression of a ricin toxin B subunit: insulin fusion protein in edible plant tissues.

Onset of juvenile Type 1 diabetes (T1D) occurs when autoreactive lymphocytes progressively destroy the insulin-producing beta-cells in the pancreatic Islets of Langerhans. The increasing lack of insulin and subsequent onset of hyperglycemia results in increased damage to nerves, blood vessels, and tissues leading to the development of a host of severe disease symptoms resulting in premature morbidity and mortality. To enhance restoration of normoglycemia and immunological homeostasis generated by lymphocytes that mediate the suppression of autoimmunity, the non-toxic B chain of the plant AB enterotoxin ricin (RTB), a castor bean lectin binding a variety of epidermal cell receptors, was genetically linked to the coding region of the proinsulin gene (INS) and expressed as a fusion protein (INS-RTB) in transformed potato plants. This study is the first documented example of a plant enterotoxin B subunit linked to an autoantigen and expressed in transgenic plants for enhanced immunological suppression of T1D autoimmunity.

Synthesis and assembly of Porphyromonas gingivalis fimbrial protein in potato tissues.

Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266-337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266-337) protein. Further, a protein band of ~160 kDa was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against periodontal disease caused by P. gingivalis.

Immunogenicity of a cholera toxin B subunit Porphyromonas gingivalis fimbrial antigen fusion protein expressed in E. coli.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.