US20140271718A1

US20140271718A1 - Novel type 1 diabetes vaccines, and methods of use

Info

Publication number: US20140271718A1
Application number: US14/216,691
Authority: US
Inventors: Joseph Larkin; Mark A. Atkinson
Original assignee: University of Florida Research Foundation Inc
Current assignee: University of Florida Research Foundation Inc
Priority date: 2013-03-15
Filing date: 2014-03-17
Publication date: 2014-09-18
Also published as: WO2014146097A1; US9700609B2; US20160022793A1

Abstract

The subject invention provides compositions for alleviating type 1 diabetes (T1D). In preferred embodiments, the compositions comprise an effective amount of one or more antigen presenting cells (APCs) that have been pulsed with one or more bacterial isolates and/or compounds from the isolates. The bacteria used to pulse the APCs are, preferably, those that confer upon the APCs the ability to inhibit the generation of diabetes-promoting T cells. In specific embodiments, these bacteria may be, for example, Eubacteria or Clostridia. In a preferred embodiment, the APCs are dendritic cells (DCs).

Description

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser. No. 61/793,321, filed Mar. 15, 2013, which is incorporated herein by reference in its entirety.

BACKGROUND

Diabetes mellitus is a family of disorders characterized by chronic hyperglycemia and the development of long-term vascular complications. This family of disorders includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other types of diabetes.
Immune-mediated (type 1) diabetes (or insulin dependent diabetes mellitus, IDDM, T1D) is a disease of children and adults for which there currently is no adequate means for prevention or cure. Type 1 diabetes, represents approximately 10% of all human diabetes. The disease is characterized by an initial leukocyte infiltration into the pancreas that eventually leads to inflammatory lesions within islets, a process called “insulitis”.
Type 1 diabetes is distinct from non-insulin dependent diabetes (NIDDM) in that only the T1D type 1 form involves specific destruction of the insulin producing beta cells of the islets of Langerhans. The destruction of beta cells appears to be a result of specific autoimmune attack, in which the patient's own immune system recognizes and destroys the beta cells, but not the surrounding alpha cells (glucagon producing) or delta cells (somatostatin producing) that comprise the pancreatic islet. The progressive loss of pancreatic beta cells results in insufficient insulin production and, thus, impaired glucose metabolism with attendant complications.
The factors responsible for T1D are complex and thought to involve a combination of genetic, environmental, and immunologic influences that contribute to the inability to provide adequate insulin secretion to regulate glycemia.
The natural history of T1D prior to clinical presentation has been extensively studied in search of clues to the etiology and pathogenesis of beta cell destruction. The prediabetic period may span only a few months (e.g., in very young children) to years (e.g., older children and adults). The earliest evidence of beta cell autoimmunity is typically the appearance of various islet autoantibodies. Metabolically, the first signs of abnormality can be observed through intravenous glucose tolerance testing (IVGTT). Later, as the disease progresses, the oral glucose tolerance test (OGTT) typically becomes abnormal. With continued beta cell destruction and frank insulinopenia, T1D becomes manifest.
Type 1 diabetes occurs predominantly in genetically predisposed persons. Concordance for T1D in identical twins is 30-50% with an even higher rate of concordance for beta cell autoimmunity, as evidenced by the presence of islet autoantibodies in these individuals (Pyke, D. A., 1979. “Diabetes: the genetic connections.” Diabetologia 17: 333-343). While these data support a major genetic component in the etiopathogenesis of T1D, environmental or non-germline genetic factors must also play important pathologic roles. Environmental factors proposed to date include viral infections, diet (e.g., nitrosamines in smoked meat, infant cereal exposure), childhood vaccines, lack of breast-feeding, early exposure to cows' milk, and aberrant intestinal functioning (Vaarala et al. 2008). Hence, while the list of potential environmental agents for T1D is large, the specific environmental trigger(s) that precipitate beta cell autoimmunity remain elusive.
Although pre-diabetogenic T cells (bearing TCR specificity for pancreatic islet cell related antigens) are essential for T1D onset, studies in rodent models (Shoda L K, et al 2005) and patients (Metcalfe K A, et al 2001; Redondo M J, et al 2001; Hyttinen V, Kaprio J, Kinnunen L, Koskenvuo M & Tuomilehto J 2003) suggest that T1D may be prevented by inhibiting their acquisition of diabetogenic effector functions. Antigen presenting cells, in particular dendritic cells, maintain immune homeostasis by providing signals sufficient to activate pathogen-specific naïve T lymphocytes while being able to induce tolerance in naïve T cells specific to self-tissues and commensal bacteria. APC modulate immune responses by providing antigen presentation, necessary co-stimulatory signals, and an appropriate cytokine environment.
A peaceful mutualism exists between resident gut bacteria and the mammals in which they reside: the host provides food for the commensal bacteria, which in turn provide nutrients to the host by metabolizing otherwise indigestible food. In addition, a dynamic equilibrium also exists between resident gut flora and the development of the mammalian immune system. In particular, Th17 effector functions are induced by resident commensal bacteria, and subsequently regulate the composition of bacteria residing within the gut (rev in (Curtis M M & Way S S 2009; and Ivanov I I, et al 2008)).
Studies have shown that modulation of gut composition can alter onset of T1D (rev. in (Vaarala O, Atkinson M A & Neu J 2008)). Moreover, it has been recently demonstrated that distinct, naturally occurring microbial communities reside within the gut of Bio-breeding diabetes prone and resistant rats (Roesch L F, et al 2009), and within the subset of female NOD mice naturally resistant to T1D compared to susceptible syngeneic mice (Kriegel M A, et al 2011).
In terms of gut microbial regulation, APC prime T lymphocyte effector functions, maintain mutualistic communities, while eliminating those perceived as pathogens. While the IL17A (referred hereafter as IL17) effector function by T lymphocytes is important in microbial gut community regulation (Happel K I, et al 2005; Higgins S C, Jarnicki A G, Lavelle E C & Mills K H 2006; Murphy C A, et al 2003), the role of APC primed IL17 production in the context of T1D, is less clear as it has been correlated with both onset and resistance (Nikoopour E, et al 2010; Bending D, et al 2009; Martin-Orozco N, Chung Y, Chang S H, Wang Y H & Dong C 2009).
Notably, increased natural segregation of gut residing Segmented Filamentous Bacteria (SFB) (Kriegel M A, et al 2011) and oral feeding of Lactobacillus johnsonii N6.2 (LjN6.2) (Valladares R, et al 2010) were sufficient to confer T1D resistance to T1D susceptible rodent strains. The resistance to T1D mediated by LjN6.2 and SFB was correlated to a Th17 bias (Kriegel M A, et al 2011; Lau K, et al 2010). Although dendritic cells prime naïve T lymphocytes and interact with resident gut flora communities directly (Grainger J R, Hall J A, Bouladoux N, Oldenhove G & Belkaid Y 2010), how distinct microbes can contribute to APC priming of diabetogenic T lymphocytes effector functions is poorly understood.
The NOD is a well-established mouse model of T1D, with destructive leukocytic infiltration of pancreatic islets, followed by insulin insufficiency in >80% of female mice. The NOR mouse, a recombinant congenic mouse strain, possesses 88% genetic identity with the NOD mouse and also develops leukocytic infiltrations within the pancreatic vasculature. However, unlike NOD mice, the leukocytic infiltrations in NOR do not typically progress to insulitis (i.e, intra-islet invasion), rendering NOR mice T1D free.
As noted above, one of the numerous factors that have been considered in the context of unraveling the complex etiology of T1D is intestinal functioning, including the interaction of intestinal microflora. The presence of a commensal intestinal microbiota in infancy is critical and well documented for numerous physiologic processes including growth, angiogenesis, optimization of nutrition, and stimulation of various arms of the innate and adaptive immune systems. However, similar studies in T1D are limited. In rodent models of T1D, the disease is likely to develop under germ free conditions. Diabetes prone rats (BB-DP) subjected to cesarean derivation develop accelerated disease (Like et al. 1991). In terms of using such information to proactively modulate diabetes formation, antibiotic treatments to BB-DP rats after weaning (Brugman et al. 2006) prevents diabetes, whereas with the NOD mouse, a decreased frequency of T1D was observed with the administration of doxycycline (Schwartz et al. 2007). Probiotic treatment of non-obese diabetic mice (NOD) prevents the onset of T1D (Calcinaro et al. 2005; Yadav et al. 2007). Similarly, a low fat diet with Lactobacillus strains reduced insulin-dependent diabetes in rats (Matsuzuki et al. 2007). Antibiotics can prevent T1D in diabetes-prone rats (BB-DP) (Brugman et al. 2006) and in NOD mice (Schwartz et al. 2006). The incidence of diabetes in NOD mice increases in a germ-free environment (Suzuki et al. 1987; Wicker et al. 1987). Freund's adjuvant, which contains mycobacteria, also protects NOD mice and the BB-DP rat against diabetes (Sadelain et al. 1990a,b; McInerney et al. 1991). The specific mechanisms of how such therapies modulate disease are unclear.
Type 1 diabetes is currently managed by the administration of exogenous human recombinant insulin. Although insulin administration is effective in achieving some level of euglycemia in most patients, it does not prevent the long-term complications of the disease including ketosis and damage to small blood vessels, which may affect eyesight, kidney function, and blood pressure and can cause circulatory system complications.
Although knowledge of the immune system has become much more extensive in recent years, the precise etiology of T1D remains a mystery. Furthermore, despite the enormously deleterious health and economic consequences, and the extensive research effort, there currently is no effective means for controlling the formation of this disease.

BRIEF SUMMARY

The subject invention provides compositions for alleviating type 1 diabetes (T1D). In preferred embodiments, the compositions comprise an effective amount of one or more antigen presenting cells (APCs) that have been pulsed with one or more bacterial isolates and/or compounds from the isolates. The bacteria used to pulse the APCs are, preferably, those that confer upon the APCs the ability to inhibit the generation of diabetes-promoting T cells. In specific embodiments, these bacteria may be, for example, Eubacteria or Clostridia. In a preferred embodiment, the APCs are dendritic cells (DCs).
The subject invention also provides methods for preventing or slowing the development of T1D. These methods comprise the administration of a composition of the subject invention, wherein the composition preferably comprises an effective amount of one or more pulsed APCs.
In accordance with the subject invention, it has been found that APCs that have been pulsed with bacteria strains can be used to alleviate (delay the onset of and/or reduce the severity or progression of), T1D. In specific embodiments of the subject invention, the administration of DCs that have been pulsed with Lactobacillus strains such as L. johnsonii can prevent or delay the onset of, or reduce the progression of, T1D in an animal model. Specially exemplified herein is the use of Lactobacillus johnsonii N6.2. Vaccination with DCs that have been pulsed with Lactobacillus johnsonii N6.2 conferred T1D resistance to DP rodents. Diabetes resistance in the DP rodents was correlated to a TH17 bias within the mesenteric lymph nodes, which was associated with high levels of IL6 and IL23.
The subject invention further provides methods to screen human gut-derived bacterial strains (in vitro) for their ability to be used in the APC vaccine described herein. In one embodiment, human gut derived bacterial strains, that have been found to be negatively correlated with diabetes onset can be incubated with human dendritic cells (obtained from blood or a human dendritic cell line) followed by assessing the ability of the gut flora modulated dendritic cell to inhibit the generation of diabetes promoting T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Resistance to T1D in NOR mice, compared to NOD, is correlated to enhanced TH17 differentiation and antigen presenting cells. A. Whole LN cells, isolated from NOD and NOR mice, were pooled and stimulated with anti-CD3. Forty-eight hours later, one half of the culture supernatant was collected from each sample and replenished with fresh media. At 72 hours each sample was pulsed with 3H thymidine, followed by assessment of proliferation by cellular incorporation of 3H thymine 16 hours later. Collected supernatants were assessed for IFNγ, IL17A, and IL6 by ELISA as indicated. Each point within graphs shown indicates an individual mouse analyzed, with experiments performed in duplicate. B. Ex vivo LN cells, isolated from NOD and NOR mice, were pooled and stained with antibodies specific for T cell and APC markers followed by analysis by flow cytometry. Top: Graphs showing absolute numbers and relative frequency of CD4+ and CD8+ lymphocytes within pooled LN from NOD and NOR mice. Bottom: Graphs showing absolute number and relative frequency of total CD11c+, CD11b+, and B220+ APC present within pooled LN cells from NOD and NOR mice. Absolute cell number counts for respective cell populations were obtained by multiplying gating frequencies obtained from flow cytometry with total cell numbers. Averages were based upon a minimum of 3 mice from each lineage **p<0.01, ***=p<0.005

FIG. 2A-2B. IL17 deficiency is correlated to decreased APC, but not CD4+CD2S+ Treg numbers. Pooled leukocyte cells isolated from the axillary, brachial, mesenteric, inguinal, and cervical LN of NOD and NOR mice were labeled with T cell and APC specific antibodies ex vivo foHowed by flow cytometry analysis. A) Graphs showing the absolute number (top) and relative frequencies (bottom) of CDS+, CD4+, and CD4+CD25+ T lymphocytes present in the LN of NOD and NOR mice. B): Graphs showing the absolute number (top) and relative frequencies (bottom) of 8220+, COI1b+, and CD11c+ leukocytes present in the LN of NOD and NOR mice. Absolute cell number counts for CD II b+, CD II c+, and B220+ populations were obtained by gating first for the CD3-population. **=p<0.01, ***=p<0.005. Data is averaged for 3 mice per set.

FIG. 3A-3B. Lymphocyte infiltrates present within the NOR pancreas display enhanced levels of TH17-related factors compared to NOD. Serial cryostat sections were obtained from the pancreases of 12 week old pre-diabetic NOD and NOR mice in order to analyze the location and phenotype of T lymphocytes infiltrates. A. H&E stains of pancreas isolated from 12 week old NOD and NOR mice. Photos are at 20× magnification and representative of 3 mice from each category. Arrows indicate representative islets present in the pancreas of each mouse lineage B. Graphs showing the relative expression of CD3, IFNγ, IL17A, RORγt, and IL6 RNA message in 40 micron cryostat sections isolated sequentially from H&E pancreas sections. Anti-CD3 results are shown relative to actin, while message levels are shown relative to anti-CD3. *=p<0.05. A total of 6 mice per category were examined in duplicate experiments.

FIG. 4A-4B. NOD mice possess reduced Th17 bias and distinct bacterial flora composition in the mesentery compared to NOR mice. A. Graphs showing the expression of CD3, IL17A, and IL23R relative to β-actin in mesenteric lymph nodes isolated from 12 week old pre-diabetic NOD and NOR mice. Each point within graphs shown indicates an individual mouse analyzed, with experiments performed in duplicate. B. Top, PCA analysis of weighted distances between NOD and NOR mice evaluating microbial diversity. Each dot represents a sample and is identified based on type. Bottom, Stacked bar graph showing the most abundant bacterial species present within stool samples isolated from NOD and NOR mice. The stacked bar graph depicts the genus of groups of bacteria sharing 97.5% sequence similarity with different abundances in NOD and NOR mice (p<0.05).

FIG. 5. Enhanced Th17 bias In NOR mesenteric LN is correlated to enhanced IL23production. Graphs showing the expression of RoRyt, IL23, and IL6 relative to f3-actin in mesenteric lymph nodes isolated from 12 week old pre-diabetic NOD and NOR mice. Each point within graphs shown indicates an individual mouse analyzed, with experiments performed in duplicate.

FIG. 6. Enhanced Th17 bias not observed in pancreatic LN. Graphs showing the expression of IL6, IL23, IL17A, and IL23R, and RORyt relative to CD3 in pancreatic lymph nodes isolated from 12 week old pre-diabetic NOD and NOR mice. Each point within graphs shown indicates an individual mouse analyzed, with experiments performed in duplicate.

FIG. 7A-7B. Analysis of diversity and microbial communities in NOD and NOR mice at the phyla and genus levels. A) Bar graph showing no difference (p=0.32) in community diversity between NOD and NOR mice based on Shannon-Weaver diversity analysis. B) Stacked bar graphs showing the most abundant bacterial communities present with stool isolated from 8 week old pre-diabetic NOD and NOR mice at the genus and phyla level.

FIG. 8A-8C. LjN6.2, but not LrTD1, increases APC activation in NOD mice. Splenocytes, derived from NOD or NOR mice, were cultured with graded doses of LjN6.2 or LrTD1 in the presence of anti-CD3. Following 48 hours incubation, samples were analyzed through flow cytometry for APC activation. A): Histograms showing changes in CD11b and CD11c frequencies upon treatment of NOD in vitro leukocyte cultures with LjN6.2 (red), LrTD1(black) or CD3 (shaded). B) Graphs showing percentage of CD11b+ and CD11c+ leukocytes in NOD and NOR cells cultures incubated with graded doses of bacteria as indicated C) Graphs showing changes in MHC expression levels on CD11c+ and CD11b+ APC upon treatment with graded doses of LjN6.2 (shaded square) or LrTD1 (open square).

FIG. 9A-9C. Enhanced dendritic cell maturation mediated by LjN6.2 surface antigens. Splenocytes, derived from NOD or NOR mice, were cultured with graded doses of LjN6.2 or LrTD1in the presence of anti-CD3, as in FIG. 4. Subsequent to incubation, samples were analyzed for expression of the endocytic marker, DEC 205. A) Overlay histograms showing changes in DEC205 frequency on CD11c+ dendritic cells upon treatment of NOD cultures with varying doses of LjN6.2 and LrTD1. B) Graphical representations of DEC205 expression on CD11c+ dendritic cells present within NOD and NOR cultures upon incubation with varying doses of LjN6.2 and LrTD1. C) BMDC, derived from NOD mice were matured in the presence of GM-CSF, followed by incubation with LjN6.2 or beadbeater lysed LjN6.2 bacterial cellular components. left, graph showing IL6 production by BMDC incubated with either intact LjN6.2, or fractional equivalents of nonviable lysed LjN6.2 components defined by ultracentrifugation as membrane (pellet components) or cytosolic (soluble) components. Right: photo showing anaerobic growth of LjN6.2 (4×105) or fractional membrane equivalent on MRS media after 48 hours. Data shown is average of at least two independent experiments depicting at least 5 individual NOD and NOR mice.

FIG. 10. Nonviable LjN6.2sU1face components mediate enhanced dendritic cell maturation. Splenocytes (4×10̂5) were isolated from C57BL/6 mice and plated in the presence of antiCD3 (4 μg/ml) and BMDC pulsed with either LjN6.2 or bacterial cellular equivalents of LjN6.2 membrane or cytosolic equivalents. Supernatants were removed at 48 hours for ELISA analysis. Graphs showing IL6 and I11? production in response to varying concentrations of LjN6.2 or cellular equivalents of membrane components as indicated.

FIG. 11. List of OTUs at 97.5% sequence similarity with different relative abundances in NOD and NOR mice and OTU correlations with cytokine message. OTUs with at least 0.1% abundance in either NOD or NOR mice and are more abundant in either mouse type (p<D.OS, indicated in parentheses) or correlated with Th 17 associated factors are listed. Positive (+) and negative (−) correlations using Spearman correlation are indicated (significant correlations alpha<0.05).

DETAILED DESCRIPTION

The subject invention provides compositions for preventing and/or delaying the onset of Type 1 Diabetes (T1D) (or reducing the severity of T1D) wherein the compositions comprise an effective amount of one or more antigen presenting cells (APCs). The composition can also include pharmaceutically acceptable carriers, additives, or excipients.
In preferred embodiments, the compositions comprise an effective amount of one or more dendritic cells (DCs) that have been primed with one or more bacterial isolates such that the primed DCs, when administered to a diabetic subject (or a subject at risk for developing diabetes), inhibit the progression of diabetes. The bacteria used to pulse the dendritic cells are, preferably, those that confer upon the DCs the ability to inhibit the generation of diabetes promoting T cells. In specific embodiments, these bacteria may be, for example, Eubacteria or Clostridia. In other embodiments, the bacteria may be Lactobacillus.
The subject invention also provides methods for preventing or slowing the development of T1D. These methods comprise the administration, to a subject with T1D or at risk for developing T1D, a composition of the subject invention, wherein the composition preferably comprises an effective amount of one or more of the primed DCs.
In accordance with the subject invention, it has been found that dendritic cells that have been pulsed with bacteria strains can be used to alleviate (delay the onset of, and/or reduce the severity or progression of), T1D. In specific embodiments of the subject invention, the administration of DCs that have been pulsed with Lactobacillus strains such as L. johnsonii can prevent or delay the onset of, or reduce the progression of, T1D in an animal model.
For example, in accordance with the subject invention, it has been found that vaccination with DCs that have been pulsed with Lactobacillus johnsonii N6.2 conferred T1D resistance to DP rodents. Diabetes resistance in the DP rodents was correlated to a TH17 bias within the mesenteric lymph nodes, which was associated with high levels of IL6 and IL23.
Lymphocytes, isolated from NOD mice, possess a reduced Th17 bias when compared to counterparts from congenic, diabetes resistant NOR mice. This is notable since NOR and NOD mice contain significant numbers of potentially diabetogenic T lymphocytes, but only NOD mice proceed to T1D. The fact that congenic, diabetes resistant NOR mice possess a Th17 bias in comparison to NOD mice is comparable to a recent study showing that a Th17 bias is present within the subset of NOD mice naturally resistant to T1D (Kriegel M A, et al 2011). Together these data suggest that in addition to the conversion of pre-diabetogenic T lymphocytes to either a Th2 lymphocyte or a Foxp3+ regulatory T cell phenotype, differentiation into the Th17 lineage may also inhibit the acquisition of diabetogenic effector functions.
In accordance with the subjection invention, diabetes inhibiting LjN6.2enhanced APC maturation as denoted by induction of Th17 lymphocytes, up-regulation of MHCI, up-regulation of MHCII, increased IL6 production, and decreased surface expression of DEC205. Additionally, we found that nonviable LjN6.2 were sufficient to mediate dendritic cell maturation, likely through an interaction between bacterial membrane components and DEC205.
The amount of the therapeutic or pharmaceutical composition of the invention that is effective in the prevention and/or treatment of T1D can be determined by a person skilled in the art having the benefit of the current disclosure through standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the practitioner and each patient's circumstances. In one embodiment, effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The subject invention further provides methods to screen human gut-derived bacterial strains (in vitro) for their ability to be used in the dendritic cell vaccine described herein. In one embodiment, human gut derived bacteria strains, that have been found to be negatively correlated with diabetes onset (using, for example, comparison techniques similar to Brown et al. 2011, which is incorporated by reference herein in its entirety), can be incubated with human dendritic cells (obtained from blood or a human dendritic cell line) followed by assessing the ability of the gut flora modulated dendritic cell to inhibit the generation of diabetes promoting T cells. Of particular interest are the microbes identified in U.S. Published Application No. US-2012-0183513-A1, which is incorporated by references in its entirety.
The bacterial strain can be a mutant having substantially the same or improved properties or it can be a naturally-occurring variant thereof Procedures for making mutants are well known in the microbiological art. Ultraviolet light and nitrosoguanidine are used extensively toward this end.
In another embodiment of the subject invention, the DCs can be pulsed with antigens or other cellular components rather than the intact cell. The antigen or other cellular component could be, for example, a cell surface molecule and would be chosen based upon the ability to confer upon the DCs the desired ability to inhibit diabetes-promoting T cells.
The DCs of the subject invention can be formulated into a vaccine composition according to known methods for preparing pharmaceutically useful compositions. Formulations are described in a number of sources, which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science (Martin E W [1995] Easton Pa., Mack Publishing Company, 19^thed.) describes formulations that can be used in connection with the subject invention. The formulations of the subject invention can include other agents conventional in the art having regard to the type of formulations described herein.
The subject invention further provides a method of preventing or slowing the development of T1D comprising administration of a composition comprising an effective amount of one or more DCs together with diet modification, and/or the administration of other therapies, including, for example, immunosuppressants.
Other autoimmune conditions to which the treatments of the subject invention may be applied include, but are not limited to, rheumatoid arthritis, multiple sclerosis, thyroiditis, Crohn's disease, inflammatory bowel disease, Addison's disease, pancreas transplantation, kidney transplantation, islet transplantation, heart transplantation, lung transplantation, and liver transplantation.

Timing of Treatment

The therapies of the subject invention can be used to alleviate type 1 diabetes.
In one embodiment, treatment is administered prior to the onset of clinical manifestation of overt type 1 diabetes. The time of administration is preferably before extensive irreversible beta cell destruction as evidenced by, for example, the clinical onset of type 1 diabetes.
As set forth in more detail below with respect to type 1 diabetes, those skilled in the art, having the benefit of the instant disclosure can utilize diagnostic assays to assess the stage of disease progression in a patient and then administer treatment at the appropriate time as set forth herein.
With regard to the early detection of type 1 diabetes, numerous autoantibodies have been detected that are present at the onset of type 1 diabetes. Also, new serologic markers associated with type 1 diabetes continue to be described. Four islet autoantibodies appear to be the most useful markers of type 1 diabetes: islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), and insulinoma-associated-2 autoantibodies (IA-2A). These are discussed in more detail below; however, the use of these markers to identify those at risk for developing type 1 diabetes is well known to those skilled in the art. In a specific embodiment of the subject invention, treatment is administered when a patient has at least one antibody marker or, preferably, at least two of the antibody markers.
ICA serve an important role as serologic markers of beta-cell autoimmunity. Seventy percent or more of Caucasians are ICA-positive at onset of type 1 diabetes. Following diagnosis, ICA frequency decreases, and fewer than 10% of patients still express ICA after 10 years. The general population frequency of ICA is between 0.1% and 0.3%. In a preferred embodiment of the subject invention, ATG is administered prior to a decrease in ICA.
IAA occur in 35-60% of children at onset of type 1 diabetes but are less common in adults. For example, in Australians with new-onset type 1 diabetes, IAA were present in 90% of children less than 5 years old, in 71% of 5-10-year-olds, and in 50% of 10-15-year-olds. In Britons with type 1 diabetes, IAA were identified in 83% of children less than 10 years old and in 56% of children 10 years old and greater.
IAA have been detected in several other autoimmune diseases. IAA were identified in 15.9% of patients with Hashimoto's thyroiditis and 13.5% of Graves' disease subjects. In another study, IAA frequencies in various thyroid autoimmune diseases were 44% in Graves' disease, 21% in primary hypothyroidism, and 23% in chronic autoimmune thyroiditis, compared with 40% in primary adrenal failure, 36% in chronic hepatitis, 40% in pernicious anemia, 25% in rheumatoid arthritis, and 29% in systemic lupus erythematosus.
Approximately 2-3% of the general population express GAD autoantibodies. These antibodies are detected in 60% or more of new-onset cases of type 1 diabetes. The IA-2A and IA-2βA general population frequencies are similar to GADA at 2-3%. IA-2A and IA-2βA are observed in 60% or more of new-onset type 1 diabetes cases.
Early biochemical evidence of beta cell injury is a decreased first-phase insulin response to the administration of intravenous glucose (IVGTT). First-phase response is defined as the insulin concentrations at +1 and +3 min following completion of an intravenous bolus injection of glucose (e.g., 0.5 g/kg). There is also a dissociation in beta cell response to secretagogues: Initially the insulin response to intravenous amino acid administration (e.g., arginine) is preserved even while first-phase responses are deficient (Ganda, O. P. et al., 1984. “Differential sensitivity to beta-cell secretagogues in early, type 1 diabetes mellitus,” Diabetes 33: 516-521). In ICA-positive individuals eventually developing insulin-dependent diabetes, first-phase insulin release diminishes at a rate of about 20-40 μU/mL/year (Srikanta, S. 1984. “Pre-type 1 diabetes, linear loss of beta cell response to intravenous glucose,” Diabetes 33: 717-720).
When beta cell mass has substantially declined to less than 50% but more than 10% of normal, the OGTT may display abnormalities such as impaired fasting glucose (110-125 mg/dL) or impaired glucose tolerance (2-h glucose post-75-g challenge: 140-199 mg/dL). An abnormal OGTT prior to the clinical onset of type 1 diabetes is more likely observed in younger children. Frank clinical diabetes usually follows within 1-2 years of the onset of oral glucose intolerance. By the time acute symptoms of type 1 diabetes develop, beta cell mass is believed to have declined by approximately 90% or more from baseline. In one embodiment of the subject invention, treatment is administered once oral glucose intolerance is observed.
Most current procedures for the prediction of type 1 diabetes involve analyses of multiple islet autoantibodies. In every such study reported, nondiabetic individuals who express combinations of islet autoantibodies are found to be at greater risk for type 1 diabetes than individuals who express fewer varieties of islet autoantibodies. In addition, the total number of types of islet autoantibodies is usually more important than the specific combination of islet autoantibodies. In type 1 diabetes subjects, islet autoantibodies can also reappear after pancreas or islet transplantation, predicting failure to become insulin-independent (Bosi, E. et al. 2001. Diabetes 50:2464-247).
Thus, in genetically predisposed individuals, an environmental trigger or triggers are believed to initiate beta cell autoimmunity, which can be identified by the presence of islet autoantibodies. With progressive beta cell damage, there is loss of first-phase insulin response to intravenous glucose administration. Subsequently the OGTT becomes abnormal, followed by symptoms of diabetes and the diagnosis of type 1 diabetes. Clearly the detection of islet autoimmunity can therefore be used as a predictive marker for the subsequent development of type 1 diabetes.
Both in nondiabetic relatives of type 1 diabetes subjects and in the general population, the detection of islet autoantibodies identifies individuals who are at high risk to develop subsequent type 1 diabetes (LaGasse, J. M. et al. 2002. Diabetes Care 25:505-511). Higher titers of ICA are more predictive than lower titers, and multiple islet autoantibodies are more powerful predictors than the presence of single autoantibodies. The combination of ICA plus low first-phase insulin secretion is possibly the strongest confirmed predictor of subsequent type 1 diabetes as demonstrated in the DPT-1. When using single autoantibodies, comparative sensitivities for the prediction of type 1 diabetes are as follows: ICA>GADA>IA-2A>>IAA. Combination islet autoantibody assays (e.g., the simultaneous detection of GADA and IA-2A (Sacks, D. B. et al. 2001. J. Clin. Chem. 47:803-804; Kawasaki, E. et al. 2000. Front Biosci. 5:E181-E190) will likely supersede ICA testing in future testing programs.
The majority of individuals with type 1 diabetes have islet autoantibodies at the time of onset of the disease. In cases where it is difficult to differentiate type1 from type 2 diabetes, the presence of one or more islet autoantibodies (e.g., ICA, IAA, GADA, or IA-2A) is diagnostic of type 1a, immune-mediated diabetes (Rubinstein, P. et al. 1981. Hum. Immunol. 3:271-275). When individuals clinically present with a subtle, non-gketotic form of diabetes that may not be insulin-requiring yet are islet autoantibody-positive, LADA is diagnosed.

Administration and Formulation of the Vaccine

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to generate an immune response. Precise amounts of cells or active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of a few thousand cells (to millions of cells) for cellular vaccines. For standard epitope or epitope delivery vaccines then the vaccine may be several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
The manner of application may vary widely; however, certain embodiments herein will most likely be delivered intravenously, subcutaneously, peritoneally, intramuscularly and vaginally or at the site of a tumor or infection directly. Regardless, any of the conventional methods for administration of a vaccine are applicable. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
In many instances, it will be desirable to have multiple administrations of the vaccine, e.g., four to six vaccinations provided weekly or every other week. A normal vaccination regimen will often occur in two to twelve week intervals or from three to six week intervals. Periodic boosters at intervals of 1-5 years, usually three years, may be desirable to maintain protective levels of the immune response or upon a likelihood of a remission or re-infection. The course of the immunization may be followed by assays for, e.g., T cell activation, cytokine secretion or even antibody production, most commonly conducted in vitro. These immune response assays are well known and may be found in a wide variety of patents and as taught herein.
The vaccine of the present invention may be provided in one or more “unit doses”. Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. The subject to be treated may also be evaluated, in particular, the state of the subject's immune system and the protection desired. A unit dose need not be administered as a single injection but may include continuous infusion over a set period of time. Unit dose of the present invention may conveniently be described in terns of DNA/kg (or protein/Kg) body weight, with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000 or more mg/DNA or protein/kg body weight being administered.
Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient. Preferably, the dosage is administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

Materials and Methods

Animals

Pre-diabetic NOD/ShiltJ and age-matched NORlltJ mice (The Jackson laboratory, Bay Harbor, Me.) were maintained in specific pathogen-free conditions at the Association for Assessment and Accreditation for laboratory Animal Care (AAALAC) accredited University of Florida, under the supervision of Institutional Animal Care and Use Committee (IACUC). Pre-diabetes status of NOD/ShiltJ mice was confirmed using a blood glucose monitoring unit (Lifescan One Touch) as having blood glucose levels below 250 mgldl. Peripheral LN (axillary, inguinal, and brachial), mesenteric LN, pancreatic LN, pancreas, and spleens were extracted from each mouse for in vitro or ex vivo analysis. Prediabetic NOD/ShiLtJ and NOR/LtJ were euthanized followed by removal of peripheral LN (axillary, inguinal, and brachial), mesenteric LN, pancreatic LN, pancreas, and spleens for in vitro or ex vivo analysis.

Bone Marrow Derived Dendritic Cell Preparation

Bone marrow was removed from the femur and tibia bones of NOD mice and washed. Progenitor cells were subsequently incubated in RPMI 1640 supplemented with 10% fetal bovine serum, 1% anti-biotic/anti-mycotic, granulocyte/macrophage colony stimulating factor (GMCSF) (20 nglml) in 24-well plates (I×106 cells/well). Old medium was removed and replaced with I ml fresh complete RPMI medium containing 20 ng/ml GM-CSF every 2 days. On day 8, aggregates were dislodged and transferred with complete RPMI medium into IOO-mm petri dishes at a maximum of I×107 cells/dish. At 24 and 48 hour time points following transfer, nonadherent, non-proliferating, maturing dendritic cells (BMDC) were collected from the dish and stored in a sterile flask.

Proliferation Assays

Lymphocyte proliferation assays were performed as previously described (1) with modifications. 4×105 whole lymphocytes were incubated with 4 ˜g1mL anti-CD3 (clone 17A2; eBioscience, San Diego, Calif. in supplemented RPMI 1640 (IO-040-CV; Cellgro, Manassas, Va.) containing 10% fetal bovine serum (10082-147; Gibeo, Carlsbad, Calif., 1% anti-biotic/anti-mycotic (30-004CI; Cellgro) in 96-well round bottom plates. After 72 hours of incubation, cultures were pulsed with 0.5 mCi eH┘ thymidine. Thymidine incorporation was measured using a Beckman LS3801 Liquid Scintillation System.

In Vitro Cytokine Secretion Analysis

Whole lymphocytes or BMOC were incubated with 4 ˜g1mL anti-CD3, L johnsonii N6.2 (LjN6.2) and/or L. reuteri TOI (LrTOI) at various concentrations as indicated. At 48 hours, 100 ˜IL of supernatant was removed from each well and replenished with fresh medium as previously described (2). Cytokine EllSAs were subsequently performed on harvested supernatants. ELISA kits were purchased from BO Bioscience: anti-IFNi′ (555138; BO Biosciences, San Diego, Calif., and anti-IL6 (555240). Capture mAb (555068) and detection mAb (555067) for IL 17A were purchased from BO Biosciences. Cytokine standard for IL-17A was purchased from eBioscience (14-8171-80).

Flow Cytometry

Single cell suspensions of pooled Lymph nodes (LN) (axillary, inguinal, brachial, mesenteric, and superficial cervical), and spleen were stained with the following mAbs for flow-cytometric analysis: anti-004-Pacific Blue (RM4-5;), anti-MHC I-FitC (KH95), anti-COI Ic-PE (N418; eBioscience, San Diego, Calif., anti-B220-APC (RA3-6B2, eBioscience), anti-COil b-A700 (M 1170), anti-CD II b-FitC (M 1170, eBioscience), anti-CD I Ie-FitC (N418, eBioscienee), anti CD86-A700 (GLI), anti-CD80-PE (16-IOAl), DEC 2OS-APC (20Syekta) and anti-MHC II-Fe (39-10-8,) mAb. All flow cytometey antibodies were purchased from BD PharMingen unless otherwise stated. 50,000-100,000 live events were collected on a LSRII (BO PharMingen) and analyzed using FlowJo software (Tree Slar, San Carlos, Calif.). The absolute numbers of cells recovered from various organs was determined by multiplying the total number of cells isolated from various tissues by the percentage of total cells bearing a lineage specific marker denoted by flow cytometey.

Pancreas RNA Isolation/Histology

Pancreas was harvested from NOD and NOR mice and snap frozen in OCT (Fisher, 14-373-65, Pittsburgh, Pa.) embedding medium in a dewer of liquid nitrogen and 2-methylbutane (Fisher, 03551-4). Blocks were sectioned on a Leica CM 1950 cryostat at a thickness of 40 microns. RNA was then isolated using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, KIT0204, Carlsbad, Calif.) and protocol. Purity of RNA was confirmed using a Nanodrop ND 1000 Spectrophotometer. Five micron sections were cut and stained with H&E. Photos were taken at 20× magnification using the Leica DM 2500 Microscope equipped with an Optronics color camera and MagnaFire software (Optronics, Goleta, Calif.).

RNA Isolation and RT-qPCR

Total RNA was extracted from the LN and spleens of NOD or NOR mice using the SV Total RNA Isolation System (promega, Corp., Madison, Wis., USA), according to the manufacturer's recommended spin column extraction protocoL The concentrations and purity of the total RNA were determined using a SmartSpecPlus Spectrophotometer (BioRad, Hercules, Calif., USA). First-strand cDNA synthesis was performed using ImProm-II Reverse Transcription System (Promega,Corp., Madison, Wis., USA) or iScript RT Supermix for RT-qPCR (BioRad, 170-8841). Absolute QPCR SYBR Green Mix (ABgene Epsom, Surrey, UK) or iQ SYBR Green Supermix Sample (BioRad, 170-8880S) and gene specific primers (Table I) al 200 nM were used to amplify relative amounts of cDNA on a PTC-2oo Peltier Thermal Cycler with a CHROMO 4 Continuous Fluorescence Detector (BioRad). Amplification was performed as previously described (Lau, 112011). The fold-change in expression was calculated using the double 8CT method (i.e. using the equation T MCT) using BioRad software.

Bacterial DNA Extraction and Analysis

Fresh stool was collected from NOD and NOR mouse strains and frozen at _—80° C. Whole DNA was extracted from each stool sample using the Qiagen DNeasy Blood and Tissue kit following the manufacturer's instructions (Qiagen, Valencia, Calif., USA). Spectrophotometry was used to determine the DNA concentration and purity of each sample. Amplification and library construction of the V4 region of bacterial J6S rRNA genes was performed using the primers 515F and 806R (3) with the addition of barcode sequences and required Illumina adapters as described in Fagen et al. (4). For the amplification, an initial denaturation step of 94° C. for 3 min, followed by 20 cycles of 94° C. for 45 sec, 50° C. for 30 sec, and 65° C. for 90 sec, and a final elongation step of 65° C. for 10 min was performed. peR products were purified using the Qiagen™ PCR purification kjt following the manufacturer's protocol (Qiagen, Valencia, Calif., USA).

Illumina Sequencing and Analysis

165 rRNA amplicon sequencing was performed using an lIlumina GAllx sequencing platform (Illumina, Inc., CA, USA) generating IOO×2 paired-end reads with an average of 60,409±17,336 reads per sample. The reads were clustered into operational taxonomic units (OTU) with 97.5% or greater similarity using USEARCH 6.0 (http://www.drive5.comlusearchl) and classified using an RDP database (RDP 10) modified by the TaxCollector program (5). Tables were created and filtered (50 reads for a given OTU in alleast 3 samples; 96.6% of reads were retained) using Lederhosen (httDs:/Igithub.com/audy/lederhosen). Bar graphs and statistical analyses were conducted in XLSTAT (version 2012.2.01; 2012 Addinsoft), an add-in to Microsoft Excel; (version 14.2.5; 2010 Microsoft Corporation, Redmond Wash.). Analyses included Spearman correlation on taxonomic (proportion of reads) and cytokine message data, PCA generation, Shannon diversity index calculation, and I-test of unequal variance to compare taxonomic abundance between NOD and NOR samples (performed using natural logtransformation of data; p-value˜0.05 was considered significant).

Mechanical Separation of Bacteria and Viability Assessment

Bacterial cells were incubated with 0.1 mm glass beads and homogenized through the use of a beadbeater (Qbiogene) according to manufacturers' instructions. Nonviable bacterial components were subsequently separated through ultracentifugation and labeled as membrane, which consisted of the pellet, and cytoplasmic, which contained the soluble fraction. Efficiency of bacterial disruption was assessed through the culture of bacteriallysates on agar plates under anaerobic conditions.

Statistical Calculations

Statistically significant differences were determined using Graph Pad Prism software using an unpaired, two-tailed student t test unless otherwise indicated. Statistical significances are indicated with asterisks symbols: *p<=0.05; **p<=0.0 I; ***p<=0.OO5
Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Diminished Peripheral Th17 Differentiation and APC Frequency is Correlated to T1D Onset

In order to better understand the relationship betweenTh17 differentiation and T1D onset, IL17 production by stimulated NOD T lymphocytes was compared to that of NOR lymphocytes. Although proliferation and IFNγ production were comparable in activated NOD and NOR peripheral LN suspensions, three-fold more IL17 was produced by NOR lymphocytes (FIG. 1A). Since APC derived IL6 is required for TH17 differentiation, its production was measured in the NOD and NOR LN suspensions. Whereas no IL6 production was detected within NOD LN suspensions, IL6 production was readily observed within the activated NOR LN suspension (FIG. 1A). Together, these data show that in vitro activated LN suspensions from diabetes resistant NOR mice possessed higher levels of Th17 differentiation than pre-diabetic NOD counterparts.
As differences in Th17 differentiation between NOD and NOR LN suspensions could be due to differences in leukocyte absolute numbers or frequencies, the cellular composition of the LN were analyzed. CD4+ and CD8+ T lymphocytes were found to be comparable in frequency and absolute number between NOD and NOR mice (FIG. 1B). Moreover, CD4+CD25+ regulatory T cells, which have been shown to play a critical role in the prevention of T1D onset, were also comparable in number and frequency between NOD and NOR peripheral LN (FIG. 2). In stark contrast, NOD LNs possessed significantly fewer APC in both number and frequency (FIG. 1B).
Although deficiencies were observed among CD11c+ dendritic cells and CD11b+ macrophages, the most significant deficiencies were observed among the B220+ B cells (FIG. S1).
Together these data suggest that the reduced capacity of NOD mice to produce IL17, in comparison to NOR, may be due in part to reductions in APC function.

EXAMPLE 2

Lymphocytes in Pancreas of NOD Mice Exhibit Reduced Th17 Bias Compared to those Present in NOR Pancreas

NOR and NOD mice experience leukocytic infiltrations of the pancreas, however NOR mice are resistant to insulitis and T1D. Since reduced amounts of IL17 were observed in the peripheral LN of NOD mice compared to NOR, differences in Th17 associated factors within the leukocytic infiltrations of the pancreas were studied. Frozen-OCT embedded sections from the pancreases of both strains were generated, which were processed to either generate H&E stains or to measure the presence of Th17 related RNA. Sequential sections were utilized so that infiltrates shown in the H&E histology could be compared to RNA message levels.
Leukocytic infiltrations were observed in pancreatic H&E stains from both pre-diabetic 12 week old NOD and age-matched NOR mice (FIG. 3A). The NOD pancreas, however, possessed significantly more T lymphocyte infiltration and profound insulitis (as denoted by H&E (FIG. 2A) and CD3 message (FIG. 3B)).
Although IFNγ levels were indistinct, significantly higher levels of Th17 associated factors RORγt and IL6 were observed in the NOR pancreatic infiltrate compared to that of the NOD (FIG. 3B). IL17 message levels were consistently higher in the NOR compared to the NOD.
Together, these data show a reduced Th17 bias within the pancreatic infiltration of pre-diabetic NOD mice compared to NOR.

EXAMPLE 3

NOD and NOR Mice Possess Distinct Th17 Biases in Mesenteric LN which is Correlated to Diverse Microbial Communities

The mesenteric LN of NOR and NOD mice were examined for distinctions in cytokine profiles.
Although mesenteric LN cells isolated from NOD mice possessed 50% more CD3 message, IL17 message levels were significantly lower in the NOD mesenteric LN (mLN) compared to the NOR. The Th17 specific transcription factor, RORγt, and surface protein, IL23 receptor (IL23R) were also consistently lower in the NOD mLN (FIG. 4A, 5). Notably, message levels for IL23 (required to sustain the Th17 phenotype), but not IL6 were consistently lower in the mLN of NOD mice compared to NOR (FIG. 5).
Although lymphocytes present in the gut preferentially track to the pancreatic LN, distinctions in Th17 associated factors were not observed within the pancreatic LN (FIG. 6).
Together, these data show that pancreas and mLN of spontaneously diabetic NOD mice, possess a significantly lower fraction of T lymphocytes bearing aTh17 bias when compared to counterpart NOR mice.
In order to analyze the bacterial communities present within NOD and NOR mice housed under the same conditions, barcoded 16S rRNA amplicon libraries were generated from stool samples obtained from the respective mice and sequenced with Illumina GAIIX. As depicted in FIG. 4B, PCA analysis revealed that the bacterial composition of NOR mice were more similar to other NOR mice than to communities obtained from NOD using 97.5% sequence similarity (depicted as weighted distances).
The Shannon diversity index detected no difference in overall community diversity between NOD and NOR mice, which was consistent with overall similarities observed at the phyla and genus levels (FIG. 7). However, 16S rRNA sequencing revealed that the abundance of specific species present in the genera Eubacterium, Clostridium, Syntrophococcus, Bacteroides,and Blautia differed significantly between the bacterial communities observed in the stool samples of NOD and NOR mice (FIG. 4B and FIG. 11).
Eubacterium strains E. dolichum and E. ventriosum were observed in higher frequency in NOD mice, while B. stercoris was 2.5 fold higher (1.0% versus 0.4%) in NOR mice. Although Clostridia were present in both NOR and NOD mice, notably distinct species were present in each (denoted Clostridia (+NOD) versus Clostridia (+NOR), FIG. 4B and Table 1). Additionally, several operational taxonomic units (OTUs) were found correlated with Th17 associated cytokine messages (FIG. 11).
Two Clostridium groups (one unidentified and another closely identifying to Clostridium alkalicellulosi) were negatively correlated with RORγt message and found in greater abundance in NOD mice, while Clostridium septicum was found to be positively correlated with IL23R message and more abundant in NOR mice. Specific Eubacterium, Ruminococcus, and Ruminofilibacter species were also correlated with Th17 factors.
Together these data suggest that the enhanced Th17 bias present within the pancreas and mesenteric LNs of diabetes resistant NOR mice, compared to NOD mice, was correlated to distinct microbial communities present within the gut.

EXAMPLE 4

Defects in Th17 Phenotype can be Reversed through APC Activation by Lactobacillus johnsonii N6.2

The oral transfer of a single strain of bacteria, LjN6.2, from diabetes resistant Bio-Breeding Diabetes Resistant (BBDR) rats to Bio-Breeding Diabetes Prone (BBDP) rats was sufficient to confer T1D resistance to BBDP rats (Valladares R, et al 2010). Moreover, the conferred T1D resistance was correlated to an APC dependent, Th17 bias observed in the BBDP rat and the NOD mouse (Lau K, et al 2011). Conversely, a second commensal bacterial strain, LrTD1, neither conferred T1D resistance nor mediated a Th17 bias (Valladares R, et al 2010; Lau K, et al 2011). The capacity of LjN6.2 to modulate APC was compared to LrTD1 in vitro. Treatment of NOD derived primary cell cultures with LjN6.2, but not LrTD1, consistently up-regulated the frequency of both CD11b+ and CD11c+ leukocytes in a dose dependent manner (FIG. 8A, B). Although the frequencies of both CD11b+ and CD11c+ leukocytes present in NOD cultures increased in a dose dependent manner, CD11b+ and CD11c+ leukocytes in NOR cultures were only moderately increased (FIG. 4B).
The capacity of LjN6.2 to modulate APC cell activation was examined. LjN6.2 treatment mediated a decrease in CD11b+MHCIhi leukocytes and an increase in the frequency CD11b+MHCIIhi leukocytes in both NOD and NOR cultures (FIG. 8C). Notably, although NOD derived CD11c+ dendritic cells, treated with LjN6.2, upregulated levels of MHC classes I and II in a dose dependent manner, LjN6.2 failed to specifically increase MHCI and MHCII expression in NOR derived CD11c+ DC (FIG. 8C). Significantly, in contrast to LjN6.2, LrTD1 treatment mediated reduced, or negligible, modulation of MHC (FIG. 8C).
Together these data show that LjN6.2, but not LrTD1, modulates the T cell priming capacity of NOD derived APC through increased frequency and up-regulation of MHC surface expression.

EXAMPLE 5

LjN6.2 Mediates Endocytic Marker, DEC205 Down-Modulation on Dendritic Cells

The capacities of LjN6.2 and LrTD1 to modulate DEC205 levels on dendritic cells was measured. LjN6.2, but not LrTD1, mediated down-regulation of DEC205 expression in both NOD and NOR CD11c+ dendritic cells in a dose dependent manner (FIG. 9). Notably, although LjN6.2 did not up regulate MHC molecules on NOR derived CD11c+ leukocytes, it did mediate DEC205 down modulation.
These data suggest that LjN6.2 interactions with DEC205 helped to mediate the capacity of dendritic cells to prime Th17 effector functions. LjN6.2 surface antigen modulates dendritic cell function.
IL6 production by BMDCs incubated with either viable LjN6.2, or bacterial cellular equivalents of bead beater ruptured LjN6.2 was measured. The bead beater ruptured LjN6.2 components were separated by centrifugation into insoluble (designated membrane) or soluble (designated cytoplasmic) components. BMDC incubated with LjN6.2 produced copious amounts of IL6 (FIG. 9C). Significantly, lysed LjN6.2 also mediated significant amounts of IL6 in a dose dependent manner (FIG. 9C and FIG. 10). In contrast, the cytoplasmic portion of the bacteria failed to up-regulate IL6 production. It is highly unlikely that the IL6 production is due to residual viable bacteria that survived the lysis process as we were unable to detect bacterial colonies from plates streaked with membrane components in contrast to plates streaked with LjN6.2 at the same initial bacterial concentration (FIG. 9C). It was also confirmed that IL6 and IL17 production could be mediated by LjN6.2 cell membrane components, but not the cytoplasmic components, in a dose dependent manner (FIG. 10).
Together, these data strongly suggest that components present on the surface of LjN6.2 specifically modulate the T cell priming capacity of dendritic cells, possibly through interactions with DEC205.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

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Claims

We claim:

1. A composition for treating or preventing type 1 Diabetes (T1D), the composition comprising an antigen presenting cell (APC) pulsed with a bacterium or a component of the bacterium, wherein the bacterium or the component of the bacterium confers upon the APC the ability to inhibit the generation of diabetes promoting T cells.

2. The composition of claim 1, wherein the APC is a dendritic cell.

3. The composition of claim 1, wherein the bacterium is selected from the genera Eubacterium, Clostridium, and Lactobacillus.

4. The composition of claim 3, wherein the bacterium is Lactobacillus johnsonii or Lactobacillus johnsonii N6.2.

5. The composition of claim 1, wherein the component of the bacterium is a membrane component.

6. The composition of claim 1, wherein the bacterium is nonviable.

7. A method of treating or preventing T1D, the method comprising administering to a subject having T1D or having an increased risk of developing T1D, a composition comprising an effective amount of an APC pulsed with a bacterium or a component of the bacterium, wherein the bacterium or the component of the bacterium confers upon the APC the ability to inhibit the generation of diabetes promoting T cells in the subject.

8. The method of claim 7, wherein the APC is DC.

9. The method of claim 7, wherein the bacterium is selected from the genera Eubacterium, Clostridium, and Lactobacillus.

10. The method of claim 7, wherein the bacterium is Lactobacillus johnsonii or Lactobacillus johnsonii N6.2.

11. The method of claim 7, wherein the component of the bacterium is a membrane component.

12. The method of claim 7, wherein the bacterium is nonviable.

13. A method of preparing a vaccine to treat or prevent T1D, the method comprising pulsing an APC with a bacterium or a component of the bacterium, wherein the bacterium or the component of the bacterium confers upon the APC the ability to inhibit the generation of diabetes promoting T cells.

14. The method of claim 13, wherein the APC is a DC.

15. The method of claim 13, wherein the bacterium is selected from the genera Eubacterium, Clostridium, and Lactobacillus.

16. The method of claim 13, wherein the bacterium is Lactobacillus johnsonii or Lactobacillus johnsonii N6.2.

17. The method of claim 13, wherein the component of the bacterium is a membrane component.

18. The method of claim 13, wherein the bacterium is nonviable.