The role of DNA methylation in the pathogenesis of type 2 diabetes mellitus.

Diabetes mellitus (DM) is a chronic condition characterised by ß cell dysfunction and persistent hyperglycaemia. The disorder can be due to the absence of adequate pancreatic insulin production or a weak cellular response to insulin signalling. Among the three types of DM, namely, type 1 DM (T1DM), type 2 DM (T2DM), and gestational DM (GDM); T2DM accounts for almost 90% of diabetes cases worldwide.Epigenetic traits are stably heritable phenotypes that result from certain changes that affect gene function without altering the gene sequence. While epigenetic traits are considered reversible modifications, they can be inherited mitotically and meiotically. In addition, epigenetic traits can randomly arise in response to environmental factors or certain genetic mutations or lesions, such as those affecting the enzymes that catalyse the epigenetic modification. In this review, we focus on the role of DNA methylation, a type of epigenetic modification, in the pathogenesis of T2DM.

The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex.

The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.

Antibody Fab-Fc properties outperform titer in predictive models of SIV vaccine-induced protection.

Characterizing the antigen-binding and innate immune-recruiting properties of the humoral response offers the chance to obtain deeper insights into mechanisms of protection than revealed by measuring only overall antibody titer. Here, a high-throughput, multiplexed Fab-Fc Array was employed to profile rhesus macaques vaccinated with a gp120-CD4 fusion protein in combination with different genetically encoded adjuvants, and subsequently subjected to multiple heterologous simian immunodeficiency virus (SIV) challenges. Systems analyses modeling protection and adjuvant differences using Fab-Fc Array measurements revealed a set of correlates yielding strong and robust predictive performance, while models based on measurements of response magnitude alone exhibited significantly inferior performance. At the same time, rendering Fab-Fc measurements mathematically independent of titer had relatively little impact on predictive performance. Similar analyses for a distinct SIV vaccine study also showed that Fab-Fc measurements performed significantly better than titer. These results suggest that predictive modeling with measurements of antibody properties can provide detailed correlates with robust predictive power, suggest directions for vaccine improvement, and potentially enable discovery of mechanistic associations.

Route of immunization defines multiple mechanisms of vaccine-mediated protection against SIV.

Antibodies are the primary correlate of protection for most licensed vaccines; however, their mechanisms of protection may vary, ranging from physical blockade to clearance via the recruitment of innate immunity. Here, we uncover striking functional diversity in vaccine-induced antibodies that is driven by immunization site and is associated with reduced risk of SIV infection in nonhuman primates. While equivalent levels of protection were observed following intramuscular (IM) and aerosol (AE) immunization with an otherwise identical DNA prime-Ad5 boost regimen, reduced risk of infection was associated with IgG-driven antibody-dependent monocyte-mediated phagocytosis in the IM vaccinees, but with vaccine-elicited IgA-driven neutrophil-mediated phagocytosis in AE-immunized animals. Thus, although route-independent correlates indicate a critical role for phagocytic Fc-effector activity in protection from SIV, the site of immunization may drive this Fc activity via distinct innate effector cells and antibody isotypes. Moreover, the same correlates predicted protection from SHIV infection in a second nonhuman primate vaccine trial using a disparate IM canarypox prime-protein boost strategy, analogous to that used in the first moderately protective human HIV vaccine trial. These data identify orthogonal functional humoral mechanisms, initiated by distinct vaccination routes and immunization strategies, pointing to multiple, potentially complementary correlates of immunity that may support the rational design of a protective vaccine against HIV.

High-resolution definition of humoral immune response correlates of effective immunity against HIV.

Defining correlates of immunity by comprehensively interrogating the extensive biological diversity in naturally or experimentally protected subjects may provide insights critical for guiding the development of effective vaccines and antibody-based therapies. We report advances in a humoral immunoprofiling approach and its application to elucidate hallmarks of effective HIV-1 viral control. Systematic serological analysis for a cohort of HIV-infected subjects with varying viral control was conducted using both a high-resolution, high-throughput biophysical antibody profiling approach, providing unbiased dissection of the humoral response, along with functional antibody assays, characterizing antibody-directed effector functions such as complement fixation and phagocytosis that are central to protective immunity. Profiles of subjects with varying viral control were computationally analyzed and modeled in order to deconvolute relationships among IgG Fab properties, Fc characteristics, and effector functions and to identify humoral correlates of potent antiviral antibody-directed effector activity and effective viral suppression. The resulting models reveal multifaceted and coordinated contributions of polyclonal antibodies to diverse antiviral responses, and suggest key biophysical features predictive of viral control.

Optimization and qualification of an Fc Array assay for assessments of antibodies against HIV-1/SIV.

The Fc Array is a multiplexed assay that assesses the Fc domain characteristics of antigen-specific antibodies with the potential to evaluate up to 500 antigen specificities simultaneously. Antigen-specific antibodies are captured on antigen-conjugated beads and their functional capacity is probed via an array of Fc-binding proteins including antibody subclassing reagents, Fcγ receptors, complement proteins, and lectins. Here we present the results of the optimization and formal qualification of the Fc Array, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. Assay conditions were optimized for performance and reproducibility, and the final version of the assay was then evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness.

Multiplexed Fc array for evaluation of antigen-specific antibody effector profiles.

Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection. However, antibody titer provides only a partial measure of antibody function, which is dependent on both the variable region (Fv) to bind the antigen target, and the constant region (Fc) to elicit an effector response from the innate arm of the immune system. In the case of some diseases, such as HIV, for which an effective vaccine has proven elusive, antibody effector function has been shown to be an important driver of monoclonal antibody therapy outcomes, of viral control in infected patients, and of vaccine-mediated protection in preclinical and clinical studies. We sought to establish a platform for the evaluation of the Fc domain characteristics of antigen-specific antibodies present in polyclonal samples in order to better develop insights into Fc receptor-mediated antibody effector activity, more fully understand how antibody responses may differ in association with disease progression and between subject groups, and differentiate protective from non-protective responses. To this end we have developed a high throughput biophysical platform capable of simultaneously evaluating many dimensions of the antibody effector response.

Correlates of Immunity to Influenza as Determined by Challenge of Children with Live, Attenuated Influenza Vaccine.

Background. The efficacy of live, attenuated live attenuated influenza vaccine(LAIV) and inactivated influenza vaccine(IIV) is poorly explained by either single or composite immune responses to vaccination. Protective biomarkers were therefore studied in response to LAIV or IIV followed by LAIV challenge in children. Methods. Serum and mucosal responses to LAIV or IIV were analyzed using immunologic assays to assess both quantitative and functional responses. Cytokines and chemokines were measured in nasal washes collected before vaccination, on days 2, 4, and 7 after initial LAIV, and again after LAIV challenge using a 63-multiplex Luminex panel. Results. Patterns of immunity induced by LAIV and IIV were significantly different. Serum responses induced by IIV, including hemagglutination inhibition, did not correlate with detection or quantitation of LAIV on subsequent challenge. Modalities that induced sterilizing immunity seen after LAIV challenge could not be defined by any measurements of mucosal or serum antibodies induced by the initial LAIV immunization. No single cytokine or chemokine was predictive of protection. Conclusions. The mechanism of protective immunity observed after LAIV could not be defined, and traditional measurements of immunity to IIV did not correlate with protection against an LAIV challenge.

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition.

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

Elite controllers (ECs) represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC) that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

λ Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response.

The role of Fc receptors in HIV prevention and therapy.

Over the past decade, a wealth of experimental evidence has accumulated supporting the importance of Fc receptor (FcR) ligation in antibody-mediated pathology and protection in many disease states. Here we present the diverse evidence base that has accumulated as to the importance of antibody effector functions in the setting of HIV prevention and therapy, including clinical correlates, genetic associations, viral evasion strategies, and a rapidly growing number of compelling animal model experiments. Collectively, this work identifies antibody interactions with FcR as important to both therapeutic and prophylactic strategies involving both passive and active immunity. These findings mirror those in other fields as investigators continue to work toward identifying the right antibodies and the right effectors to be present at the right sites at the right time.

Enhancing the Quality of Antibodies to HIV-1 Envelope by GagPol-Specific Th Cells.

The importance of Fc-dependent effector functions of Abs induced by vaccination is increasingly recognized. However, vaccination of mice against HIV envelope (Env) induced a skewed Th cell response leading to Env-specific Abs with reduced effector function. To overcome this bias, GagPol-specific Th cells were harnessed to provide intrastructural help for Env-specific B cells after immunization with virus-like particles containing GagPol and Env. This led to a balanced Env-specific humoral immune response with a more inflammatory Fc glycan profile. The increased quality in the Ab response against Env was confirmed by FcγR activation assays. Because the Env-specific Th cell response was also biased in human vaccinees, intrastructural help is an attractive novel approach to increase the efficacy of prophylactic HIV Env-based vaccines and may also be applicable to other particulate vaccines.

Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys.

Preclinical studies of viral vector-based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys.

Bioenergetics of the spinal cord in experimental autoimmune encephalitis of rats.

BACKGROUND: Mitochondrial dysregulation is important in axonal damage and demyelination in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). There is however, no evidence in the literature of any study that has examined cellular bioenergetics of the central nervous system (CNS) during the early development and clinical course of EAE. EAE, a rodent model of relapsing/remitting MS, is a CD4(+) T cell-mediated disease of the CNS. We hypothesize that CNS bioenergetics might predict prognosis, and that preserved bioenergetics might underlie the remission from disease. The study aims therefore, to determine whether the clinical history of EAE is influenced by cellular respiration of the CNS in susceptible Dark Agouti (DA) and resistant Albino Oxford (AO) rats. METHODS: Experimental autoimmune encephalomyelitis was induced by myelin basic protein in complete Freud Adjuvant in the footpads of DA and AO rats. A phosphorescence analyzer that determines cellular respiration was used to monitor oxygen consumption and ATP concentration was measured using the Enliten ATP assay system. Disease pathology was demonstrated by H&E and Luxol fast blue staining of sections of the lumbar regions of the spinal cord. Mitochondrial size in relation to axonal size was determined by electron microscopy. Apoptosis was studied by HPLC measurement of intracellular caspase-3 activity and caspase immunohistochemistry. Role and source of caspase 1 was studied by double immunofluorescence with antibodies for caspase-1, microglia (anti-Iba1) and astrocytes (anti-GFAP). RESULTS: The cellular respiration of the CNS did not vary between diseased and normal rats. We also demonstrate here, that at the peak of disease, inflammation as shown by caspase-1, produced by activated microglia and infiltrating cells, was significant in susceptible DA rats. The mitochondrial:axonal size ratio did not vary in the different groups although mitochondria were smaller in spinal cords of diseased DA rats. Demyelination, observed only in areas of mononuclear infiltration of the spinal cord of diseased DA rats, was demonstrated by light microscopy and electron microscopy. CONCLUSION: We conclude that EAE at this early stage does not significantly affect CNS cellular respiration and this might underlie the reason for the recovery of diseased rats.

Microscale purification of antigen-specific antibodies.

Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain.

Live attenuated and inactivated influenza vaccines in children.

BACKGROUND: Live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) are available for children. Local and systemic immunity induced by LAIV followed a month later by LAIV and IIV followed by LAIV were investigated with virus recovery after LAIV doses as surrogates for protection against influenza on natural exposure. METHODS: Fifteen children received IIV followed by LAIV, 13 an initial dose of LAIV, and 11 a second dose of LAIV. The studies were done during autumn 2009 and autumn 2010 with the same seasonal vaccine (A/California/07/09 [H1N1], A/Perth/16/09 [H3N2], B/Brisbane/60/08). RESULTS: Twenty-eight of 39 possible influenza viral strains were recovered after the initial dose of LAIV. When LAIV followed IIV, 21 of 45 viral strains were identified. When compared to primary LAIV infection, the decreased frequency of shedding with the IIV-LAIV schedule was significant (P = .023). With LAIV-LAIV, the fewest viral strains were recovered (3/33)--numbers significantly lower (P < .001) than shedding after initial LAIV and after IIV-LAIV (P < .001). Serum hemagglutination inhibition antibody responses were more frequent after IIV than LAIV (P = .02). In contrast, more mucosal immunoglobulin A responses were seen with LAIV. CONCLUSIONS: LAIV priming induces greater inhibition of virus recovery on LAIV challenge than IIV priming. The correlate(s) of protection are the subject of ongoing analysis. CLINICAL TRIALS REGISTRATION: NCT01246999.

Highly parallel characterization of IgG Fc binding interactions.

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.

Pam3CSK(4) enhanced beta cell loss and diabetogenesis: the roles of IFN-gamma and IL-17.

Toll like receptors are primary sensors of both innate and adaptive immune systems. They activate APCs and influence T-cell function in inflammatory autoimmune response. Studies have shown that TLR manipulation may lead to either tolerance or trigger autoimmunity. Using diabetogenic and subdiabetogenic multiple low doses of streptozotocin, we demonstrate here that Pam3 CYS-CK4 a TLR-2 agonist, enhances and promotes diabetes in C57BL/6 male mice following increased apoptosis of ß islet cells. FACS analysis of isolated pancreatic lymph node cells revealed significant increased number of macrophages, dendritic cells, CD4(+) TNF-α(+), CD4(+) IFN-γ(+) and most significantly, CD4(+) IL-17(+) and reduced number of CD25(+)Fox p3(+) T cells after Pam3CSK4 treatment. Genetic deletion of IFN-γ prevents whereas deletion of IL-17 reduced severity of Pam3CSK4-induced enhancement of diabetes. TLR-2 agonist-enhanced diabetogenesis is also influenced by enhanced influx of antigen presenting cells and suppression of regulatory T cell activity.