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Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with SARS-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two MERS-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate infection of human gut cells, but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that addition of exogenous trypsin also rescues replication of HKU5-CoV, a second MERS-like group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate emergence potential of CoVs and offer a means to recover previously unrecoverable zoonotic CoV strains.ImportanceOverall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

Background More and more studies focus on the relationship between the gastrointestinal microbiome and type 2 diabetes, but few of them have actually explored the relationship between enterotypes and type 2 diabetes. Materials and Methods. We enrolled 134 patients with type 2 diabetes and 37 nondiabetic controls. The anthropometric and clinical indices of each subject were measured. Fecal samples of each subject were also collected and were processed for 16S rDNA sequencing. Multiple logistic regression analysis was used to determine the associations of enterotypes with type 2 diabetes. Multiple linear regression analysis was used to explore the relationship between lipopolysaccharide levels and insulin sensitivity after adjusting for age, BMI, TG, HDL-C, DAO, and TNF-α. The correlation analysis between factors and microbiota was identified using Spearman correlation analysis. The correlation analysis between factors was identified using partial correlation analysis. Results Gut microbiota in type 2 diabetes group exhibited lower bacterial diversity compared with nondiabetic controls. The fecal communities from all subjects clustered into two enterotypes distinguished by the levels of Bacteroides and Prevotella. Logistic regression analysis showed that the Bacteroides and Bacteroides and Prevotella enterotype. Partial correlation analysis showed that lipopolysaccharide was closely associated with diamine oxidase, tumor necrosis factor-alpha, and Gutt insulin sensitivity index after adjusting for multiple covariates. Furthermore, the level of lipopolysaccharide was found to be an independent risk factor for insulin sensitivity. Conclusions We identified two enterotypes, Bacteroides and Prevotella, among all subjects. Our results showed that the Bacteroides enterotype was an independent risk factor for type 2 diabetes, which was due to increased levels of lipopolysaccharide causing decreased insulin sensitivity.Bacteroides and Prevotella enterotype. Partial correlation analysis showed that lipopolysaccharide was closely associated with diamine oxidase, tumor necrosis factor-alpha, and Gutt insulin sensitivity index after adjusting for multiple covariates. Furthermore, the level of lipopolysaccharide was found to be an independent risk factor for insulin sensitivity. Bacteroides and

Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation., Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea, emergence and spread of viral variants that harbored mutations in the RBD, D510G and I529T, was observed. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. In this study, we investigated whether they also exerted proviral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Neither mutation modulated S protein binding to sialic acids, S protein activation by host cell proteases, or inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and sera from MERS patients. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4. IMPORTANCE MERS-CoV has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted proviral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor DPP4.

Infection with Middle East respiratory syndrome coronavirus (MERS-CoV) could be asymptomatic or cause mild influenza-like illness. Therefore, the prevalence of MERS-CoV infections in the general population could be underestimated, which necessitates active surveillance to determine the epidemiological importance of asymptomatic cases. The aim of this study is to evaluate the performance of various serological assays and to estimate the seroprevalence of anti-MERS-CoV antibodies in high- and low-risk groups in Qatar. A total of 4858 samples were screened, including 4719 samples collected from healthy blood donors (BD) over a period of five years (2012-2016), 135 samples from baseline case contacts (CC) collected from individuals in close contact with three positive PCR-confirmed patients (CP), and four samples from MERS-CoV CP. Initial screening using anti-MERS-CoV IgG (IgG rS1-ELISA kit) revealed ten reactive samples from BD (10/4719, 0.21%), one from CC (1/135, 0.74%), and three from CP (3/4, 75%). Samples from CP but not from BD were also reactive by whole-virus anti-MERS-CoV IgG (n=3/4) and IgM (n=1/4) indirect immunefluorescent tests (IIFT) and pseudoparticle neutralization test (ppNT). The reactive sample from CC was also confirmed by ppNT. Surprisingly, one out of thirteen (7.7%) randomly selected IgG rS1-ELISA-negative BD samples from the initial screening was reactive by the IgM-IIFT (but not by the IgG-IIFT) and was subsequently confirmed by ppNT. All IgG rS1-ELISA-reactive samples from BD exhibited considerable reactivity to the four circulating human coronaviruses (HKU1, OC43, 229E, and NL63). Cross-reactivity with SARS was only reported for samples from CP using IgG and IgM-IIFT. In conclusion, we report a low prevalence of anti-MERS antibodies in the general population, which coincides with the low number of all reported cases by the time of our study (2017) in Qatar (n=21). The false-positive results and the observed cross-reactivity between MERS-CoV and other circulating human coronavirus necessitate more detailed evaluation of available serological assays.

MERS-CoV is present in dromedary camels throughout the Middle East and Africa. Dromedary camels are the primary zoonotic reservoir for human infections. Interruption of the zoonotic transmission chain from camels to humans, therefore, may be an effective strategy to control the ongoing MERS-CoV outbreak. Here we show that vaccination with an adjuvanted MERS-CoV Spike protein subunit vaccine confers complete protection from MERS-CoV disease in alpaca and results in reduced and delayed viral shedding in the upper airways of dromedary camels. Protection in alpaca correlates with high serum neutralizing antibody titers. Lower titers of serum neutralizing antibodies correlate with delayed and significantly reduced shedding in the nasal turbinates of dromedary camels. Together, these data indicate that induction of robust neutralizing humoral immune responses by vaccination of naïve animals reduces shedding that potentially could diminish the risk of zoonotic transmission.

Gene based delivery of immunoglobulins promises to safely and durably provide protective immunity to individuals at risk of acquiring infectious diseases such as HIV. We used a rhesus macaque animal model to optimize delivery of naturally-arising, autologous anti-SIV neutralizing antibodies expressed by Adeno-Associated Virus 8 (AAV8) vectors. Vectored transgene expression was confirmed by quantitation of target antibody abundance in serum and mucosal surfaces. We tested the expression achieved at varying doses and numbers of injections. Expression of the transgene reached a saturation at about 2 x 1012 AAV8 genome copies (gc) per needle-injection, a physical limitation that may not scale clinically into human trials. In contrast, expression increased proportionately with the number of injections. In terms of anti-drug immunity, anti-vector antibody responses were universally strong, while those directed against the natural transgene mAb were detected in only 20% of animals. An anti-transgene antibody response was invariably associated with loss of detectable plasma expression of the antibody. Despite having atypical glycosylation profiles, transgenes derived from AAV-directed muscle cell expression retained full functional activity, including mucosal accumulation, in vitro neutralization, and protection against repeated limiting dose SIVsmE660 swarm challenge. Our findings demonstrate feasibility of a gene therapy-based passive immunization strategy against infectious disease, and illustrate the potential for the nonhuman primate model to inform clinical AAV-based approaches to passive immunization., Author summary Antibodies are the humoral component of an immune response against an invading pathogen or vaccine immunogen. For challenging vaccine targets, as an alternative to active vaccination to induce the immune system to generate antibodies, current research is exploring the delivery of these proteins to populations at high risk of infection as prophylactics against infectious diseases, like HIV, RSV, and Ebola, amongst others. Passive vaccination via purified protein will require periodic reinjection to retain protective levels in subjects, adding a barrier to large scale coverage. Alternatively, delivery of antibodies using gene therapy may provide a one-time passive vaccination alternative. This strategy comes with its own hurdles, including anti-vector immunity, anti-drug immunity, physical limitations of vector uptake and the need to confirm antibody functionality. To date, many passive vaccinations strategies remain untested in humans. Non-human primate models of infection are frequently useful for predicting the success of vaccine candidates or concepts. Here, we characterize and optimize a rhesus macaque model for the delivery of anti-viral antibodies via the gene therapy vector adeno-associated virus. Lastly, we demonstrate the ability of the mAbs to protect against viral challenge. Our work demonstrates the feasibility and utility of vectored delivery of antibody transgenes in rhesus macaques. We hope this model of antibody delivery may be applied to various disease models in non-human primates and will inform clinical trial design of passive vaccination against infectious diseases.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.

Lipotoxic injury of pancreatic β cells is an important pathological feature in type 2 diabetes mellitus (T2DM). Stimulator of interferon genes (STING) can recognize its own DNA leaked into the cytoplasm from damaged mitochondria or nuclei of the host cell, thus activating its downstream factor interferon regulatory factor 3 (IRF3), causing inflammation and apoptosis. The STING-IRF3 signaling pathway is closely related to glycolipid metabolism, but its relationship with the lipotoxicity of pancreatic β cells has rarely been reported. Here, we investigated the role of the STING-IRF3 signaling pathway in lipotoxicity-induced inflammation, apoptosis, and dysfunction of pancreatic β cells. We examined the activation of STING and IRF3 in islets of db/db mice and identified the role of the STING-IRF3 signaling pathway in palmitic acid (PA)-induced lipotoxic injury of INS-1, a rat insulinoma cell line. STING and phosphorylated IRF3 including downstream interferon-β were upregulated in islets of db/db mice and PA-induced INS-1 cells. Gene silencing of STING or IRF3 ameliorated PA-induced INS-1 cell inflammation and apoptosis, and reversed impaired insulin synthesis. Additionally, PA induced downregulation of the phosphoinositide 3-kinase-AKT signaling pathway, and impaired high glucose-stimulated insulin secretion was reversed after knockdown of STING or IRF3. Our results suggest that activation of the STING-IRF3 pathway triggers inflammation and apoptosis of pancreatic β cells, leading to β-cell damage and dysfunction. Hence, inhibition of this signaling pathway may represent a novel approach for β-cell protection in T2DM.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and—in half the subjects—multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.

The role of inflammation in pathogenesis of diabetic retinopathy (DR) is getting increasingly recognized. However, it is unclear whether and how non-proliferative diabetic retinopathy (NPDR) is affected by Interleukin-17A (IL-17A) and Interleukin-22 (IL-22), two well-known inflammatory factors, and irisin, a novel potential anti-inflammatory factor. Here we recruited 40 type 2 diabetes mellitus (T2DM) patients with NPDR, 60 T2DM patients without DR (no-DR), and 20 normal glucose tolerance (NGT) controls. Serum levels of IL-17A, IL-22, and irisin were examined. Compared with NGT and no-DR subjects, NPDR group had significantly higher IL-17A levels. Irisin levels were significantly lower in T2DM patients, while IL-22 levels were not significantly different across all three groups. Multiple logistic regression analysis revealed that IL-17A significantly increased the risk of NPDR (OR = 1.22, P

A delayed predator-prey model with disease in the predator and stage structure for the prey is investigated. By analyzing the corresponding characteristic equations, the local stability of each of feasible equilibria is studied. The existence of Hopf bifurcations at the disease-free equilibrium and the coexistence equilibrium are addressed, respectively. By using Lyapunov functions and LaSalle invariant principle, sufficient conditions are derived for the global stability of the trivial equilibrium, the predator-extinction equilibrium and the disease-free equilibrium, respectively. Further, sufficient conditions are derived for the global attractiveness of the coexistence equilibrium of the proposed system.

Summary Understanding how broadly neutralizing antibodies (bnAbs) to influenza hemagglutinin (HA) naturally develop in humans is critical to the design of universal influenza vaccines. Several classes of bnAbs directed to the conserved HA stem were found in multiple individuals, including one encoded by heavy-chain variable domain VH6-1. We describe two genetically similar VH6-1 bnAb clonotypes from the same individual that exhibit different developmental paths toward broad neutralization activity. One clonotype evolved from a germline precursor recognizing influenza group 1 subtypes to gain breadth to group 2 subtypes. The other clonotype recognized group 2 subtypes and developed binding to group 1 subtypes through somatic hypermutation. Crystal structures reveal that the specificity differences are primarily mediated by complementarity-determining region H3 (CDR H3). Thus, while VH6-1 provides a framework for development of HA stem-directed bnAbs, sequence differences in CDR H3 junctional regions during VDJ recombination can alter reactivity and evolutionary pathways toward increased breadth., Graphical Abstract, Highlights • Two VH6-1 influenza HA stem-specific clonotypes are isolated from one individual • Precursors for these two VH6-1 clonotypes bind different influenza A HA groups • Differences in the CDRH3 conformation mediate the distinctive binding profiles • Somatic hypermutation leads to similar binding breadth between the two clonotypes, Wu et al. describes two genetically similar influenza antibody clonotypes from the same individual. Clonotype precursors recognize different influenza A HA groups, but both develop cross-group neutralizing capability through affinity maturation. Structural characterization reveals that the specificity difference between clonotypes is mainly due to conformational variation in CDR H3.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection and continues to infect humans, thereby contributing to a high mortality rate (34.3% in 2019). In the absence of an available licensed vaccine and antiviral agent, therapeutic human antibodies have been suggested as candidates for treatment. In this study, human monoclonal antibodies were isolated by sorting B cells from patient's PBMC cells with prefusion stabilized spike (S) probes and a direct immunoglobulin cloning strategy. We identified six receptor-binding domain (RBD)-specific and five S1 (non-RBD)-specific antibodies, among which, only the RBD-specific antibodies showed high neutralizing potency (IC50 0.006-1.787 μg/ml) as well as high affinity to RBD. Notably, passive immunization using a highly potent antibody (KNIH90-F1) at a relatively low dose (2 mg/kg) completely protected transgenic mice expressing human DPP4 against MERS-CoV lethal challenge. These results suggested that human monoclonal antibodies isolated by using the rationally designed prefusion MERS-CoV S probe could be considered potential candidates for the development of therapeutic and/or prophylactic antiviral agents for MERS-CoV human infection.

The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.

A stage-structured predator–prey model with a transmissible disease spreading in the predator population and a time delay due to the gestation of the predator is formulated and analyzed. By analyzing corresponding characteristic equations, the local stability of each feasible equilibria and the existence of Hopf bifurcations at the disease-free equilibrium and the coexistence equilibrium are addressed, respectively. By using Lyapunov functions and the LaSalle invariant principle, sufficient conditions are derived for the global stability of the trivial equilibrium, the predator–extinction equilibrium and the disease-free equilibrium, respectively. Further, sufficient conditions are derived for the global attractiveness of the coexistence equilibrium of the proposed system. Numerical simulations are carried out to support the theoretical analysis.

β-Cell insufficiency plays an important role in the development of diabetes. Environmental factors, including lifestyle, play a critical role in β-cell dysfunction. Modern lifestyles affect the inherent circadian clock in central and peripheral organs. Recent studies have demonstrated that the normal intrinsic circadian clock in islets was essential for the viability of β cells and their insulin secretory function. Overall, however, the data are inconclusive. Our study demonstrated that the disrupted circadian rhythm of islets in streptozotocin induced type1 diabetic mice may be associated with impaired β-cell function and glucose intolerance. Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, could partially restore the normal circadian rhythm and activate the 5' AMP-activated protein kinase (AMPK) signaling pathway. Our study provided evidence demonstrating that Liraglutide might restore β-cell function and protect against the development of diabetes in a mouse model by attenuating the disruption of the intrinsic circadian rhythm in islets and by activating AMPK signaling.

The association of blood glucose [including fasting blood glucose (FBG), 2-h postload blood glucose, and glycated hemoglobin (HbA1c)] with the risk of a mild decline in the estimated glomerular filtration rate (eGFR) in healthy subjects was unclear. The aim of the study was to investigate this association in middle-aged and elderly healthy Chinese. The study included 1,112 healthy Chinese who were ≥40 years old, and all the subjects were divided into two groups based on FBG value of 5.6 mmol/L. A mildly reduced eGFR was defined as 60–90 mL/min/1.73 m2. Multiple linear or logistic regression analysis was used to estimate the association of blood glucose with eGFR and the risk of a mildly reduced eGFR, respectively. A generalized additive model was used to explore a possible nonlinear relationship between FBG and eGFR. FBG was significantly associated with decreased eGFR and increased risk of a mildly reduced eGFR independent of age, gender, body mass index, waist circumference, systolic blood pressure (BP), diastolic BP, triglyceride, high-density lipoprotein cholesterol, fasting insulin, smoking, and drinking. Additionally, FBG and eGFR showed a nonlinear association (P

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the “out” position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections. IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

Background Islet dysfunction and destruction are the common cause for both type 1 and type 2 diabetes mellitus (T2DM). The islets of Langerhans are highly vascularized miniorgans, and preserving the structural integrity and full function of the microvascular endothelium is vital for protecting the islets from the infiltration of immune cells and secondary inflammatory attack. Mesenchymal stromal cell (MSC)-based therapies have been proven to promote angiogenesis of the islets; however, the underlying mechanism for the protective role of MSCs in the islet endothelium is still vague. Methods In this study, we used MS-1, a murine islet microvascular endothelium cell line, and an MSC-MS1 transwell culturing system to investigate the protective mechanism of rat bone marrow-derived MSCs under oxidative stress in vitro. Cell apoptosis was detected by TUNEL staining, annexin V/PI flow cytometry analysis, and cleaved caspase 3 western blotting analysis. Endothelial cell activation was determined by expression of intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as well as eNOS phosphorylation/activation. The changes of VCAM-1, eNOS, and the β-catenin expression were also tested in the isolated islets of T2DM rats infused with MSCs. Results We observed that treating MS-1 cells with H2O2 triggered significant apoptosis, induction of VCAM expression, and reduction of eNOS phosphorylation. Importantly, coculturing MS-1 cells with MSCs prevented oxidative stress-induced apoptosis, eNOS inhibition, and VCAM elevation in MS-1 cells. Similar changes in VCAM-1 and eNOS phosphorylation could also be observed in the islets isolated from T2DM rats infused with MSCs. Moreover, MSCs cocultured with MS-1 in vitro or their administration in vivo could both result in an increase of β-catenin, which suggested activation of the β-catenin-dependent Wnt signaling pathway. In MS-1 cells, activation of the β-catenin-dependent Wnt signaling pathway partially mediated the protective effects of MSCs against H2O2-induced apoptosis and eNOS inhibition. Furthermore, MSCs produced a significant amount of Wnt4 and Wnt5a. Although both Wnt4 and Wnt5a participated in the interaction between MSCs and MS-1 cells, Wnt4 exhibited a protective role while Wnt5a seemed to show a destructive role in MS-1 cells. Conclusions Our observations provide evidence that the orchestration of the MSC-secreted Wnts could promote the survival and improve the endothelial function of the injured islet endothelium via activating the β-catenin-dependent Wnt signaling in target endothelial cells. This finding might inspire further in-vivo studies.

Background Nonalcoholic fatty liver disease (NAFLD) is a common result of obesity and metabolic syndrome. Hepatocyte injury and metabolic disorders are hallmarks of NAFLD. Stimulator of interferon genes (STING) and its downstream factor interferon regulatory factor 3 (IRF3) trigger inflammatory reaction in response to the presence of cytosolic DNA. STING has recently been shown to play an important role in early alcoholic liver disease. However, little is known about the role of STING-IRF3 pathway in hepatocyte injury. Here, we aimed to examine the effect of STING-IRF3 pathway on hepatocyte metabolism, inflammation and apoptosis. Methods We examined the activation of the STING-IRF3 pathway, a high-fat diet (HFD)-induced obese mouse model, and determined the role of this pathway in a free fatty acid (FFA)-induced hepatocyte inflammatory response, injury, and dysfunction in L-O2 human liver cells. Results STING and IRF3 were upregulated in livers of HFD-fed mice and in FFA-induced L-O2 cells. Knocking down either STING or IRF3 led to a significant reduction in FFA-induced hepatic inflammation and apoptosis, as evidenced by modulation of the nuclear factor κB (NF-κB) signaling pathway, inflammatory cytokines, and apoptotic signaling. Additionally, STING/IRF3 knockdown enhanced glycogen storage and alleviated lipid accumulation, which were found to be associated with increased expression of hepatic enzymes in glycolysis and lipid catabolism, and attenuated expression of hepatic enzymes in gluconeogenesis and lipid synthesis. Conclusions Our results suggest that the STING-IRF3 pathway promotes hepatocyte injury and dysfunction by inducing inflammation and apoptosis and by disturbing glucose and lipid metabolism. This pathway may be a novel therapeutic target for preventing NAFLD development and progression.

Irisin, a newly identified myokine responsible for browning of white or beige adipocytes, has been reported to be present at reduced levels in diabetic patients and associated with obesity, serum triglyceride (TG) levels, and intrahepatic TG levels. We wondered whether irisin could directly affect fatty acid and TG metabolism in adipocytes and hepatocytes. We examined the effects of various concentrations of irisin on lipolysis (according to Oil Red O staining, free fatty acid release, and glycerol release), protein expression of HSL and ATGL, and mRNA expression of other lipid-related genes (UCP-1, PPARγ, FABP-4, HSL, ATGL, PPARα, and CPT-1) in mature 3T3-L1 adipocytes, as well as mRNA levels of genes involved in the synthesis (SREBP-1C and FAS) and β-oxidation (PPARα and CPT-1) of fatty acids in HepG2 hepatocytes under physiological or hyperglycemic conditions. Our results revealed that although irisin significantly increased the mRNA levels of UCP-1 and PPARα, it failed to show detectable effects on lipolysis, HSL or ATGL protein levels, or the mRNA expression of other lipid-related genes in mature 3T3-L1 adipocytes. In HepG2 hepatocytes, high glucose induced the upregulation of SREBP-1C and FAS and the downregulation of PPARα; however, no significant effect of irisin on gene expression was observed under either physiological or hyperglycemic conditions. We therefore conclude that irisin has no significant direct effect on lipolysis in 3T3-L1 adipocytes or on fatty acid metabolism in HepG2 hepatocytes.

The global properties of a delayed predator-prey model with Holling type II functional response and stage structure for both the predator and the prey are investigated. By analyzing the corresponding characteristic equations, the local stability of each of feasible equilibria of the model is discussed. Further, it is proved that the model undergoes a Hopf bifurcation at the coexistence equilibrium. By means of the persistence theory on infinite dimensional systems, it is shown that the model is permanent if the coexistence equilibrium exists. By using Lyapunov functions and the LaSalle invariant principle, it is shown that the trivial equilibrium is globally asymptotically stable when the predator-extinction equilibrium is not feasible, the predator-extinction equilibrium is globally asymptotically stable when the coexistence equilibrium is not feasible, and sufficient conditions are derived for the global stability of the coexistence equilibrium. Numerical simulations are carried out to illustrate the main results.

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell–mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. In this study we show low seroreactivity in humans against simian- (sAd11, sAd16) or chimpanzee-derived (chAd3, chAd63) compared with human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype, and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107–109 particle units), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least, as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFN-γ+TNF-α+IL-2+ and KLRG1+CD127−CD8+ T cells, but strikingly ∼30–80% of memory CD8+ T cells coexpressed CD127 and KLRG1. To further optimize CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ∼60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared with prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively.

Antibodies that bind to both H1 and H3 influenza strains exist in the pre-vaccination serum repertoire of healthy adults; most vaccine-elicited clonotypes bind either H1 or H3 strains. Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2∗02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2∗02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Objective. To investigate changes in the glycated hemoglobin A1c (A1c) level and those inβcell function and insulin resistance in newly diagnosed and drug naive type 2 diabetes patients and to evaluate the relationship between them.Design and Methods. A total of 818 newly diagnosed diabetic individuals who were ≥40 years of age were recruited. The subjects were grouped by A1c values (βcell function (HOMA-β) and insulin resistance (HOMA-IR). ANOVA,t-tests, and binary logistic regression analysis were used for data analysis.Results. Compared with subjects with A1c values βindex. However, the HOMA-βindex was significantly decreased at A1c values ≥7% and further decreased by 9.3% and by 23.7%, respectively, at A1c values of 7-8% and 8-9%. As A1c increased to ≥9%, a 62% reduction inβcell function was observed, independently of age, gender, body mass index (BMI), blood pressure (BP), blood lipids, and hepatic enzyme levels. Meanwhile, insulin resistance was significantly increased with an increase in A1c values.Conclusions. Elevated A1c values (≥7%) were associated with substantial reductions inβcell function.

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.

Based on integrated pest management, a stage-structured predator-prey system with Holling type-II functional response concerning impulsive control strategy is investigated. By the Floquet theory and small amplitude perturbation skills, it is proved that there exists a globally stable pest-eradication periodic solution when the impulsive period is less than some critical values. Further, sufficient conditions for the permanence of the system is established. Numerical simulations are carried out to illustrate the effect of impulses on the dynamics of the system.

A stage-structured predator-prey system with time delay and Beddington- DeAngelis functional response is considered. By analyzing the corresponding characteristic equation, the local stability of a positive equilibrium is investigated. The existence of Hopf bifurcations is established. Formulae are derived to determine the direction of bifurcations and the stability of bifurcating periodic solutions by using the normal form theory and center manifold theorem. Numerical simulations are carried out to illustrate the theoretical results.

A predator–prey model with time delay and Holling type-II functional response is investigated. By choosing time delay as the bifurcation parameter and analyzing the associated characteristic equation of the linearized system, the local stability of the system is investigated and Hopf bifurcations are established. The formulae determining the direction of bifurcations and the stability of bifurcating periodic solutions are given by using the normal form theory and center manifold theorem. Numerical simulations are carried out to illustrate the theoretical results.

A stage-structured predator-prey system with time delay is considered. By analyzing the corresponding characteristic equation, the local stability of a positive equilibrium is investigated. The existence of Hopf bifurcations is established. Formulae are derived to determine the direction of bifurcations and the stability of bifurcating periodic solutions by using the normal form theory and center manifold theorem. Numerical simulations are carried out to illustrate the theoretical results. Based on the global Hopf bifurcation theorem for general functional differential equations, the global existence of periodic solutions is established.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and systemic immune responses by different immunization routes, alone or in combination with rAd5. rAd41 expressing HIV envelope (Env) protein induced cellular immune responses comparable to those of rAd5-based vectors after either a single intramuscular injection or a DNA prime/rAd boost. Oral priming with rAd41-Env followed by intramuscular boosting with rAd5-Env stimulated a more potent CD8 + T-cell response in the small intestine than the other immunization regimens. Furthermore, the direct injection of rAd41-Env into ileum together with intramuscular rAd5-Env boosting increased Env-specific cellular immunity markedly in mucosal as well as systemic compartments. These data demonstrate that heterologous rAd41 oral or ileal priming with rAd5 intramuscular boosting elicits enhanced intestinal mucosal cellular immunity and that oral or ileal vector delivery for primary immunization facilitates the generation of mucosal immunity.

We report optical planar waveguide formation and modal characterization in β-BaB2O4 crystals by Cu2+-ion implantation at an energy of 3.0 MeV and doses of ∼ 1014 ions/cm2. The prism-coupling method was used to investigate the dark-mode property at wavelengths of 633 nm and 1539 nm. The refractive-index profile of the waveguide was reconstructed by an effective refractive index method. The modal analysis indicates that the fields of TM modes can be well restricted in the guiding region, which means the formation of a non-leaky waveguide in the crystal. The results show that the β-BaB2O4 waveguides may be used in the application of high efficiency frequency conversion.

Macrophages are important targets for HIV-1, and R5X4 strains play a central role in pathogenesis, especially in late-stage patients who may receive the fusion inhibitor T20 (enfuvirtide). Sensitivity to T20 varies markedly among HIV-1 strains and is influenced by viral and cellular factors that affect Env/CD4/coreceptor interactions. We addressed the relation between T20 inhibition and the pathway by which R5X4 HIV-1 infects primary macrophages, which express both coreceptors. In U87/CD4/coreceptor cells, T20 sensitivity for entry through CCR5 and CXCR4 was correlated. In macrophages, the proportion of total entry mediated by each coreceptor differed among isolates. Neither pathway was uniformly more or less sensitive to T20, however, nor did the proportion of entry mediated by each coreceptor predict T20 sensitivity. T20 sensitivity for macrophage infection overall correlated modestly with that for entry through CCR5 but not through CXCR4; however, unlike U87 cells, sensitivity of entry through CCR5 and CXCR4 was not correlated. These results suggest that strain-specific factors influence R5X4 T20 sensitivity regardless of the coreceptor used, an absence of systematic differences in efficiency by which R5X4 strains use the 2 coreceptors, and that efficiency and kinetics of interactions with CCR5 are central determinants of macrophage entry even when both pathways are utilized.

Betatrophin and irisin are two recently identified hormones which may participate in regulating pancreaticβ-cell function. However, the associations of these two hormones withβ-cell function remain unclear. The present study aims to demonstrate the associations of circulating betatrophin and irisin levels withβ-cell function, assessed by the area under the curve (AUC) of C-peptide, and the possible correlation between these two hormones in previously diagnosed type 2 diabetes mellitus (T2DM) patients. In total, 20 age-, sex-, and body mass index- (BMI-) matched normal glucose tolerance (NGT) subjects and 120 previously diagnosed T2DM patients were included in this study. Partial correlation analysis was used to evaluate the relationships between these two hormones and indexes ofβ-cell function and insulin resistance. Our results showed that betatrophin levels were significantly elevated, while irisin levels were significantly decreased, in patients with T2DM compared with NGT subjects. However, partial correlation analysis showed that betatrophin levels did not correlate withβ-cell function-related variables or insulin resistance-related variables before or after controlling multiple covariates, while irisin correlated positively with insulin sensitivity but is not associated withβ-cell function-related variables. Besides, no correlation was observed between betatrophin and irisin levels. Hence we concluded that betatrophin and irisin were not associated withβ-cell function in previously diagnosed T2DM patients.

Type 1 diabetes is a T cell-mediated organ-specific autoimmune disease. Antigen-specific immune intervention allows the selective targeting of autoreactive T cell, while leaving the remainder of the immune system intact. However, immune intervention for type 1 diabetes has not yielded perfect results clinically. In our paper published previously, we asked whether pancreatic duodenal home box 1 (PDX1) is a target of anti-islet autoimmunity in type 1 diabetes. In this experiment, we assessed the therapeutic effect of oral administration of PDX1 on diabetes development of 4-week-old non-obese diabetic (NOD) mice. The results indicate that PDX1 immunization is an effective intervention strategy for delaying the onset of diabetes in NOD mice in association with: 1) reduced insulitis; 2) suppression of destructive autoreactive T cells; 3) augmentation of regulatory T cells; 4) a shift in cytokine production. The present observations suggest that immunization with PDX1 modulates immune cell responses in NOD mice, raising the possibility that it is beneficial in ameliorating autoimmune destruction of beta-cells and delaying type 1 diabetes development clinically.

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development., Unmet need exists for a vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV). Here the authors report the establishment and evaluation, in mice and primates, of a series of MERS-CoV immunogens and show that they can serve as promising leads for vaccine development.

Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development.

Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in nonhuman primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, although immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 μg/ml, although immune suppression with cyclosporine (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against simian-human immunodeficiency virus (SHIV) infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled. IMPORTANCE Sustained interventions that can prevent HIV-1 infection are needed to halt the spread of the HIV-1 pandemic. The protective capacity of anti-HIV antibody gene therapy has been established in mouse models of HIV-1 infection but has not been established for primates. We show here a proof-of-concept that gene transfer of anti-HIV antibody genes can protect against infection by viruses that cause AIDS in primates when host immune responses are controlled.

Objective C-peptide has been reported to be a marker of subclinical atherosclerosis in type 2 diabetes mellitus (T2DM) patients, whereas its role in coronary artery disease (CAD) has not been clarified, especially in diabetics with differing body mass indices (BMIs). Design and Methods This cross-sectional study included 501 patients with T2DM. First, all subjects were divided into the following two groups: CAD and non-CAD. Then, binary logistic regression was used to determine the risk factors for CAD for all patients. To clarify the role of obesity, we re-divided all subjects into two additional groups (obese and non-obese) based on BMI. Finally, binary logistic regression was used to determine the risk factors for CAD for each weight group. Results The patients with CAD showed a higher BMI and fasting C-peptide level in addition to an increased prevalence of traditional risk factors for CAD, such as hypertension, insulin resistance, higher cholesterol, cysteine-C (Cys-C) and lower estimated glomerular filtration rate (eGFR). Logistic regression analysis showed that fasting C-peptide (OR=1.513, p=0.005), insulin treatment (OR=1.832, p=0.027) hypertension (OR=1.987, p=0.016) and hyperlipidemia (OR=4.159, p

Recently we demonstrated that attachment of three copies of murine C3d (muC3d) to the E2 envelope protein of bovine viral diarrhea virus results in a 10,000-fold increase in the immunogenicity of E2. Here we describe the cloning of the bovine homolog of C3d (boC3d), construction of an E2-boC3d expression cassette and expression and purification of the E2-boC3d fusion protein. We then show that E2, when coupled to boC3d, exhibits greatly enhanced immunogenicity. Thus, boC3d represents the second mammalian C3d homolog, thus far, shown to enhance the immunogenicity of a protein to which it has been coupled. Although the primary sequence of boC3d differs from muC3d by about 19%, we were able to demonstrate the enhanced immunogenicity of E2-boC3d using mice. The ability of boC3d to function in mice provides a less costly and more convenient animal model than cattle for the preliminary evaluation of E2-boC3d and other bovine antigen-boC3d fusion proteins.

The use of DNA and protein subunit vaccines in animals provides an opportunity to introduce vaccines that are arguably the safest that can be developed. For that reason, considerable effort is under way to devise methods of enhancing the immunogenicity of such vaccines. Seven years ago it was shown that fusing complement fragment C3d to hen egg lysozyme (HEL) enhanced the immunogenicity of HEL 10,000-fold. Based on this observation, we decided to evaluate the effect of C3d on the immunogenicity of the E2 protein of bovine viral diarrhea virus (BVDV). E2 is the major target of neutralizing antibody during BVDV infection. To test the effect of C3d on E2 immunogenicity, expression cassettes encoding a secreted form of E2 alone (E2s) or E2 fused to three copies of murine C3d (E2s-C3d) were constructed. The proteins were purified from the supernatants of transfected cells and used to immunize mice. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for E2s-specific antibody and by a virus neutralization test. The ELISA results indicated that the E2s-C3d protein is 10,000-fold more immunogenic than the E2s protein alone. The maximum primary immune response was elicited with

Recently, the possibility of using virus vectors to immunize cattle against selected bovine viral diarrhea virus (BVDV) genes has gained widespread interest. However, when we attempted to express the E2 protein from type 2 (890 strain) BVDV in a bovine herpesvirus 1 (BHV1) vector, we observed that expression was poor. This often happens when genes from a cytoplasmic virus are expressed in the cell nucleus. To counter this effect, we attempted to enhance expression by a strategy employed by viruses. RNAs of retroviruses and hepadnaviruses contain cis -acting elements that facilitate expression of RNAs that otherwise are degraded or retained within the nucleus. In Mason-Pfizer monkey virus, the required RNA sequence element is known as a constitutive transport element (CTE). A related element from woodchuck hepatitis virus is known as the woodchuck posttranscriptional regulatory element (WPRE). We tested the ability of the CTE, the WPRE, and introns to enhance expression of E2. All three elements stimulated expression of E2 from plasmids. The combination of the WPRE and an intron yielded the highest level of E2 expression in plasmids. However, when E2 was expressed from a BHV1 vector, the presence of an intron was inhibitory. In contrast, the WPRE was very efficient at stimulating E2 expression from a BHV1 vector. This result represents the first expression of a type 2 BVDV E2 protein from a mammalian virus vector and raises the possibility that the WPRE may provide a general method of enhancing foreign gene expression from BHV1 and other herpesvirus vectors.

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle with a world-wide distribution. Recently, the possibility of using recombinant virus vectors to immunize cattle against selected BVDV genes has gained widespread interest. Among the virus vectors tested, bovine herpesvirus-1 (BHV1) provides many unique advantages. However, results of recent studies have raised the possibility that the codon usage pattern required for optimal expression in a BHV1-infected cell may be incompatible with the codon usage pattern of BVDV. If true, use of BHV1 to express BVDV proteins would require construction of synthetic BVDV genes that have been modified to resemble the codon pattern of BHV1. To explore this possibility, we constructed a BHV1 recombinant containing the genomic form of the BVDV (NADL) E2 ORF and compared expression of the E2 protein with that of the endogenous BHV1 gD protein. We observed that E2 was expressed at a significant rate compared to that of the gD protein. We conclude that codon usage problems are unlikely to constitute a serious problem for expression of BVDV proteins in BHV1 vectors.

Insulin resistance (IR) plays an important pathophysiological role in the development of diabetes, dyslipidemia, hypertension, and cardiovascular disease. Moreover, IR can occur even in non-obese people without diabetes. However, direct detection of IR is complicated. In order to find a simple surrogate marker of IR early in non-obese people, we investigate the association of commonly-used biochemical markers (liver enzymes and lipid profiles) with IR in urban middle-aged and older non-obese Chinese without diabetes.This cross-sectional study included 1 987 subjects (1 473 women). Fasting blood samples were collected for measurement of glucose, insulin, liver enzymes, lipid profiles and creatinine. Subjects whose homeostasis model of assessment-IR (HOMA-IR) index values exceeded the 75th percentile (2.67 for women and 2.48 for men) of the population were considered to have IR. The area under the receiver operating characteristic curve (ROC) was used to compare the power of potential markers in identifying IR.Triglycerides (TG) and ratio of TG to high-density lipoprotein cholesterol (TG/HDL-C) discriminated IR better than other indexes for both sexes; areas under the receiver operating characteristic (ROC) curves (AUC) values were 0.770 (95% confidence interval 0.733-0.807) and 0.772 (0.736-0.809), respectively, for women and 0.754 (0.664-0.844) and 0.756 (0.672-0.840), respectively, for men. To identify IR, the optimal cut-offs for TG and TG/HDL-C ratio were 1.315 mmol/L (sensitivity 74.3%, specificity 71.0%) and 0.873 (sensitivity 70.1%, specificity 73.4%), respectively, for women, and 1.275 mmol/L (sensitivity 66.7%, specificity 74.4%) and 0.812 (sensitivity 75.8%, specificity 69.2%), respectively, for men.TG and TG/HDL-C ratio could be used to identify IR in urban middle-aged and older non-obese Chinese without diabetes.

The role of obesity as a determinant of kidney dysfunction has not reached an agreement and the underlying reason may be due to the heterogeneity of obese phenotypes. The aim of the study was to explore the associations of different obese phenotypes with the change of estimated glomerular filtration rate (eGFR) and the roles of obesity and metabolic abnormalities in this association. eGFR was calculated in 8,586 participants (≥40 years old). eGFR 60–90 mL/min/1.73 m2 was defined as the mildly reduced eGFR. Multiple logistic regression analysis was used to determine odds ratios (ORs) for mildly reduced eGFR in the metabolically healthy obese (MHO), metabolically abnormal non-obese (MANO) and metabolically abnormal obese (MAO) groups, using the metabolically healthy non-obese (MHNO) subjects as the reference group. Meanwhile, the associations of body mass index (BMI), waist circumference (WC) and metabolic abnormalities (including hypertension, hyperglycemia and dyslipidemia) with the risk of mildly reduced eGFR were also investigated. The proportion of MHNO, MHO, MANO and MAO subjects was 8.3, 17.1, 10.1 and 64.5 %, respectively. Increased ORs were observed in MANO (OR 1.51, P = 0.014) and MAO (OR 1.47, P = 0.015) groups, after adjusting for age, gender, smoking, drinking, BMI and WC. When further adjusting for metabolic abnormalities, MANO (OR 1.24, P = 0.247) and MAO (OR 1.17, P = 0.366) subjects would not present increased risk of mildly reduced eGFR any more. Oppositely, fasting insulin (OR 1.03, P

ObjectiveTo investigate how the glucose variability between fasting and a 2-h postload glucose state (2-h postload plasma glucose [2hPG]-fasting plasma glucose [FPG]) is associated with chronic kidney disease (CKD) in middle-aged and elderly Chinese patients previously diagnosed with type 2 diabetes.Design and methodsThis cross-sectional study included 1054 previously diagnosed type 2 diabetes patients who were 40 years of age and older. First, the subjects were divided into two groups based on a glycated hemoglobin (HbA1c) value of 7%. Each group was divided into two subgroups, with or without CKD. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to estimate the glomerular filtration rate (GFR). CKD was defined as eGFRResultsIn the patients with HbA1c levels ≥7%, the 2hPG-FPG was significantly associated with decreased eGFR and an increased risk of CKD independent of age, gender, body mass index (BMI), systolic blood pressure (BP), diastolic BP, smoking, and drinking, as well as fasting insulin, cholesterol, triglyceride, and HbA1c levels. The patients with 2hPG-FPG values ≥144 mg/dl showed an increased odds ratio (OR) of 2.640 (P = 0.033). Additionally, HbA1c was associated with an increased risk of CKD in patients with HbA1c values ≥7%.ConclusionsThe short-term glucose variability expressed by 2hPG-FPG is closely associated with decreased eGFR and an increased risk of CKD in patients with poor glycemic control (HbA1c≥7%).