TCS01 Two-Component System Influenced the Virulence of Streptococcus pneumoniae by Regulating PcpA.

Streptococcus pneumoniae relies on two-component systems (TCSs) to regulate the processes of pathogenicity, osmotic pressure, chemotaxis, and energy metabolism. The TCS01 system of S. pneumoniae is composed of HK01 (histidine kinase) and RR01 (response regulator). Previous studies have reported that an rr01 mutant reduced the pneumococcal virulence in rat pneumonia, bacteremia, a nasopharyngeal model, and infective endocarditis. However, the mechanism of TCS01 (HK/RR01) regulating pneumococcal virulence remains unclear. Here, pneumococcal mutant strains Δrr01, Δhk01, and Δrr01&hk01 were constructed, and bacterial adhesion and invasion to A549 cells were compared. RNA sequencing was performed in D39 wild-type and Δrr01 strains, and transcript profile changes were analyzed. Differentially expressed virulence genes in the Δrr01 strain were screened out and identified by quantitative real-time PCR (qRT-PCR). Our results showed that pneumococcal mutant strains exhibited attenuated adhesion and invasion to A549 cells and differential transcript profiles. Results of qRT-PCR identification showed that the differential virulence genes screened out were downregulated. Among those changed virulence genes in the Δrr01 strain, the downregulated expression level of choline binding protein pcpA was the most obvious. Complementation of rr01 and overexpression of pcpA in the Δrr01 strain partially restored both pneumococcal adhesion and invasion, and rr01 complementation made the expression of pcpA upregulated. These findings revealed that rr01 influenced pneumococcal virulence by regulating pcpA.

Reversible Three-Color Fluorescence Switching of an Organic Molecule in the Solid State via "Pump-Trigger" Optical Manipulation.

In photoswitches that undergo fluorescence switching upon ultraviolet irradiation, photoluminescence and photoisomerization often occur simultaneously, leading to unstable fluorescence properties. Here, we successfully demonstrated reversible solid-state triple fluorescence switching through "Pump-Trigger" multiphoton manipulation. A novel fluorescence photoswitch, BOSA-SP, achieved green, yellow, and red fluorescence under excitation by pump light and isomerization induced by trigger light. The energy ranges of photoexcitation and photoisomerization did not overlap, enabling appropriate selection of the multiphoton light for "pump" and "trigger" photoswitching, respectively. Additionally, the large free volume of the spiropyran (SP) moiety in the solid state promoted reversible photoisomerization. Switching between "pump" and "trigger" light is useful for three-color tunable switching cell imaging, which can be exploited in programmable fluorescence switching. Furthermore, we exploited reversible dual-fluorescence switching in a single molecular system to successfully achieve two-color super-resolution imaging.

Deciphering the Carrier Transport Properties in Two-Dimensional Perovskites via Surface-Enhanced Raman Scattering.

2D layered organic-inorganic perovskites have attracted substantial attention due to their high stability and promising optoelectronic properties. However, in-depth insights on the anisotropic carrier transport properties of these 2D perovskites are remaining challenging, while they are significant for further designing the high-performance device applications. Here, the carrier transport properties within 2D perovskite single crystals are investigated and a layered-carrier-transport model is developed through the non-invasive and non-destructive surface-enhanced Raman scattering techniques. The carrier transport features of 2D perovskites show clearly the thickness-, applied voltage- and anisotropy-dependent behaviors, which are demonstrated to origin from the quantum confinement effect. The findings elucidate the carrier transport mechanisms within 2D perovskites from their molecular level through Raman spectroscopy, thus providing a promising way for exploring the photo-physical properties in wide-ranged halide perovskites and designing highly efficient perovskite optoelectronic devices.

Conjugation of Hemoglobin and Mannan Markedly Improves the Immunogenicity of Domain III of the Zika Virus E Protein: Structural and Immunological Study.

Zika virus (ZIKV) leads to congenital microcephaly and anomalies and severe neurological diseases such as Guillain-Barre syndrome. Safe and effective vaccines are necessitated to deal with these severe health threats. As an ideal antigen, the domain III of the envelope protein (EDIII) of ZIKV can evoke potent neutralizing antibodies without any antibody-dependent enhancement (ADE) effect. However, EDIII necessitates to be formulated with an antigen delivery system or adjuvants to improve its immunogenicity. Hemoglobin (Hb) regulates inflammation, cytokine levels, and activate macrophage. Mannan is a polysaccharide of the fungal cell wall with an immunomodulatory activity. In this study, EDIII was conjugated with Hb and mannan, using the disulfide bond as the linker. Hb and mannan both functioned as the adjuvants. Conjugation of Hb and mannan acted as the delivery system for EDIII. The structure of EDIII was essentially maintained upon conjugation of Hb and mannan. The intracellular release of EDIII from the conjugate (HM-EDIII-2) was achieved by reduction of the glutathione-sensitive disulfide bond. As compared with EDIII, HM-EDIII-2 elicited high EDIII-specific IgG titers and high levels of Th1-type cytokines (IFN-γ and IL-2) and Th2-type cytokines (IL-5 and IL-10), along with no apparent toxicity to the organs. Moreover, the pharmacokinetic study revealed a prolonged serum exposure of HM-EDIII-2 to the immune cells. Thus, HM-EDIII-2 could boost a strong humoral and cellular immune response to EDIII. Our study was expected to provide the feasibility necessary to develop a robust and potentially safe ZIKV vaccine.

Analysis of laser plasma dynamics using the time resolved nonlinear optical response of ablated carbon nanocomposites mixed with epoxy resin.

Nonlinear optical properties of carbon nanostructures attract attention due to the unique response of these materials during interactions with ultrashort laser pulses. Here we probe the carbon nanocomposites mixed with epoxy resin in laser-induced plasmas using the high-order harmonics generation (HHG) method. We analyze the nanosecond pulses induced plasmas containing three carbon nanostructures (fullerenes, multiwalled carbon nanotubes and diamond nanoparticles) using 40 fs pulses propagating through these plasmas. HHG efficiencies in ablated graphite and nanocomposites are compared. We utilize two digitally synchronized (nanosecond and femtosecond) laser sources allowing for the HHG-based analysis of the evolution of different plasma plumes up to 10 µs delay from the beginning of ablation. The role of different carbon-containing nanocomposites is analyzed and the evidence for the presence of various nanomaterials in laser-induced plasma at the moment of propagation of the driving femtosecond pulses is demonstrated.

Resonance-enhanced high harmonic in metal ions driven by elliptically polarized laser pulses.

Resonance enhancement of a single order harmonic has been a main attractive feature in high-harmonic generation from laser ablated plumes of metals. Although it has been extensively investigated experimentally and theoretically, studies so far have focused only on linearly polarized driving fields. In this Letter, we study the dependence of the resonant harmonic yield in tin ions on the driving laser ellipticity. We find that the resonance leads to a less rapid decay of the harmonic yield as a function of driving ellipticity, and it is qualitatively reproduced by quantum mechanical simulations. To the best of our knowledge, our findings provide a new type of evidence for supporting previously proposed mechanisms for enhancement.

Negative Regulation of SIRT1 by IRF9 Involved in Hyperlipidemia Acute Pancreatitis Associated with Kidney Injury.

BACKGROUND: Interferon regulatory factor 9 (IRF9) acts as a negative regulator of sirtuin-1 (SIRT1) to participate in many diseases. However, the role of SIRT1 and IRF9 in hyperlipidemia acute pancreatitis associated with kidney injury is unclear. AIMS: To explore the function of SIRT1 and IRF9 in hyperlipidemia acute pancreatitis associated with kidney injury and provide theoretical guidance for disease diagnosis and treatment. METHODS: Model rats were established by intraperitoneal injection of 20% L-arginine. Apoptosis of kidney tissue was determined by TUNEL staining. Expressions of IRF9, SIRT1, p53, and acetylated p53 were detected by qRT-PCR and Western blot. Dual-Luciferase Reporter Assay was carried out to validate the regulation of IRF9 on SIRT1. RESULTS: Pancreatic and renal injury was more serious, and apoptosis of kidney epithelial cells increased in acute pancreatitis (AP) and hyperlipidemia acute pancreatitis (HLAP) group. IRF9, p53, and acetylated p53 were up-regulated, and SIRT1 was down-regulated in AP and HLAP group (p < 0.05). Down-regulation of SIRT1 was negatively correlated with up-regulation of IRF9 in AP and HLAP group (p < 0.05). Pancreatic and renal injury and kidney epithelial cells apoptosis in HLAP group were more obvious than AP group (p < 0.05). The up-regulation of IRF9 and down-regulation of SIRT1 in HLAP group were more than AP group (p < 0.05). The promoter activity of SIRT1 was repressed by IRF9. CONCLUSION: In pancreatitis associated with kidney injury, IRF9 was a negative regulator of SIRT1, down-regulated the expression of SIRT1, increased acetylated p53, and promoted renal cell apoptosis. Hyperlipidemia further aggravated pancreatic and renal injury and renal cell apoptosis.

The combination treatment of cholesterol-loaded methyl-ß-cyclodextrin and methyl-ß-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-ß-cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl-ß-cyclodextrin (MßCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38-39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MßCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-ß-cyclodextrin and methyl-ß-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

A Highly Sensitive Single Crystal Perovskite-Graphene Hybrid Vertical Photodetector.

Organolead trihalide perovskites have attracted significant attention for optoelectronic applications due to their excellent physical properties in the past decade. Generally, both grain boundaries in perovskite films and the device structure play key roles in determining the device performance, especially for horizontal-structured device. Here, the first optimized vertical-structured photodetector with the perovskite single crystal MAPbBr3 as the light absorber and graphene as the transport layer is shown. The hybrid device combines strong photoabsorption characteristics of perovskite and high carrier mobility of flexible graphene, exhibits excellent photoresponse performance with high photoresponsivity (≈1017.1 A W-1 ) and high photodetectivity (≈2.02 × 1013 Jones) in a low light intensity (0.66 mW cm-2 ) under the actuations of 3 V bias and laser irradiation at 532 nm. In particular, an ultrahigh photoconductive gain of ≈2.37 × 103 is attained because of fast charge transfer in the graphene and large recombination lifetime in the perovskite single crystal. The vertical architecture combining perovskite crystal with highly conductive graphene offers opportunities to fulfill the synergistic effect of perovskite and 2D materials, is thus promising for developing high-performance electronic and optoelectronic devices.

Boosting Perovskite Photodetector Performance in NIR Using Plasmonic Bowtie Nanoantenna Arrays.

Triple-cation mixed metal halide perovskites are important optoelectronic materials due to their high photon to electron conversion efficiency, low exciton binding energy, and good thermal stability. However, the perovskites have low photon to electron conversion efficiency in near-infrared (NIR) due to their weak intrinsic absorption at longer wavelength, especially near the band edge and over the bandgap wavelength. A plasmonic functionalized perovskite photodetector (PD) is designed and fabricated in this study, in which the perovskite ((Cs0.06 FA0.79 MA0.15 )Pb(I0.85 Br0.15 )3 ) active materials are spin-coated on the surface of Au bowtie nanoantenna (BNA) arrays substrate. Under 785 nm laser illumination, near the bandedge of perovskite, the fabricated BNA-based plasmonic PD exhibits ≈2962% enhancement in the photoresponse over the Si/SiO2 -based normal PD. Moreover, the detectivity of the plasmonic PD has a value of 1.5 × 1012 with external quantum efficiency as high as 188.8%, more than 30 times over the normal PD. The strong boosting in the plasmonic PD performance is attributed to the enhanced electric field around BNA arrays through the coupling of localized surface plasmon resonance. The demonstrated BNA-perovskite design can also be used to enhance performance of other optoelectronic devices, and the concept can be extended to other spectral regions with different active materials.

Conjugation of ß-Glucan with the Hydrazone and Disulfide Linkers Markedly Improves the Immunogenicity of Zika Virus E Protein.

The diseases caused by Zika virus (ZIKV) have received widespread concerns. As a key viral element of ZIKV, E protein was an ideal antigen for vaccine development. However, the poor immunogenicity of E protein necessitated the formulation with adjuvants. Formulation of E protein by conjugation with ß-glucan was a strategy to improve the immunogenicity of E protein, where ß-glucan was a polysaccharide adjuvant that could activate macrophages and trigger intracellular processes. However, the antigenic epitopes of E protein and the immunomodulatory sites of ß-glucan were shielded in the conjugate. Moreover, the conjugate might elicit the undesired immune response to ß-glucan. Thus, the acidic-labile hydrazone and the thiol-sensitive disulfide bonds were used as the linkers between E protein and ß-glucan. Hydrazone hydrolysis and disulfide reduction could sufficiently detach the two components in the immune cells to overcome the two disadvantages. As compared with the conjugate without the two linkers, the conjugate with the two linkers (E-PS-4) elicited high E protein-specific IgG titers and low ß-glucan-specific IgG titers. E-PS-4 elicited high levels of IFN-γ, TNF-α, IL-2, and IL-10. Moreover, E-PS-4 greatly facilitated the activation of dendritic cells without significant toxicity to the organs. A pharmacokinetic study revealed that the serum duration of E-PS-4 was longer than that of E protein. Accordingly, conjugation of E protein with ß-glucan by the hydrazone and disulfide linkers could promote a potent cellular and humoral immune response to E protein. Thus, our study could facilitate the development of an effective vaccine against ZIKV.

A novel function of IRF9 in acute pancreatitis by modulating cell apoptosis, proliferation, migration, and suppressing SIRT1-p53.

Acute pancreatitis (AP) is an inflammatory disease caused by the abnormal activation of pancreatic enzymes in the pancreas, with a considerably high morbidity and mortality. However, the etiological factor and pathogenesis of AP are still unclear. This study was aimed to explore the role and mechanism of interferon regulatory factor 9 (IRF9) in the occurrence of AP and to provide experimental and theoretical foundation for AP diagnosis and treatment. AP model in vitro was established by caerulein-induced group. Small interfering RNA (siRNA) was designed and constructed to silence IRF9 gene. After siRNA transfected and caerulein treated successfully, the expression levels of IRF9, SIRT1, and acetylated p53 (Ac-p53) were determined by qRT-PCR and Western blot. The apoptosis, proliferation, and migration of AR42J cells were checked by flow cytometry, MTT, and transwell assay. Dual-luciferase reporter assay was implemented to validate the regulatory effect of IRF9 on SIRT1. Here, our study showed that the expression of IRF9 and Ac-p53 was increased, SIRT1 was decreased, and cell apoptosis, proliferation, and migration of AR42J cells were increased after caerulein induced. IRF9 gene silencing upregulated SIRT1, downregulated Ac-p53, and inhibited cell apoptosis, proliferation, and migration. Dual-Luciferase reporter assay showed that IRF9 could negatively regulate SIRT1. The potential mechanism was that IRF9 could modulate cell apoptosis, proliferation, migration, and bind the promoter of SIRT1 to repress SIRT1-p53. It hinted that IRF9 showed a novel function in AP by modulating cell apoptosis, proliferation, migration, and suppressing SIRT1-p53. IRF9 might be a good potential treatment target for AP.

Atomic Force Microscopy to Identify Dehydration Temperatures for Small Volumes of Active Pharmaceutical Ingredients.

The environmental conditions associated with changing the hydration state of active pharmaceutical ingredients (API) are crucial to understanding their stability, bioperformance, and manufacturability. Identifying the dehydration event using < 1µg of material is an increasingly important challenge. Atomic Force Microscopy indentation mapping is implemented at controlled temperatures between 25-100°C, for nanoscale volumes of hydrated APIs exhibiting distinct dehydration behavior and anhydrous APIs as controls. For caffeine hydrate and azithromycin dihydrate, the relative mechanical modulus increases ~10-fold at dehydration temperatures. These are confirmed by conventional macroscopic measurements including Variable Temperature Powder X-ray Diffraction, Thermogravimetric Analysis, and Differential Scanning Calorimetry. Conversely, no such mechanical transition is observed for anhydrous ibuprofen or a proprietary anhydrous compound. AFM-based mechanical mapping is therefore demonstrated for small-volume determination of temperature-induced solid-state dehydration events, which may enable spatially or temporally mapping for future studies of dehydration mechanisms and kinetics, as a function of commercially relevant nanoscale heterogeneities.

Split aptamer-based detection of adenosine triphosphate using surface enhanced Raman spectroscopy and two kinds of gold nanoparticles.

An ultrasensitive and highly selective method is described for the determination of adenosine triphosphate (ATP) via surface-enhanced Raman scattering (SERS). Two split aptamers are used for specific recognition of ATP. They were attached to two SERS substrates. The first was placed on a nanolayer of gold nanoparticle-decorated graphene oxide (GO/Au3), and the other on gold nanoparticles (Au2). When ATP is introduced, it will interact with the split aptamers on the gold nanostructures to form a sandwich structure that brings the GO/Au3 nanolayer and the Au2 nanoparticle in close proximity. Consequently, the SERS signal, best measured at 1072 cm-1, is strongly enhanced. The sandwich structure also displays good water solubility and stability. Under optimized conditions, the SERS signal increases in the 10 pM - 10 nM ATP concentration range, and the limit of detection (LOD) is 0.85 pM. The method was applied to the determination of ATP in spiked human serum, and the LODs in serum and buffer are comparable. In our perception, the method has a wide scope in that numerous other aptamers may be used. This may result in a variety of other highly sensitive aptasensors for use in in-vitro diagnostics. Graphical abstract Schematic presentation of a self-assembly sandwich nanostructure as unique SERS assay platform for the sensitive detection of ATP.

Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase.

Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (∼6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas it significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK, and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.

The phenyl linker markedly increases the immunogenicity of the pneumococcal polysaccharide conjugate vaccine.

OBJECTIVE: Capsular polysaccharide (PS) of Streptococcus pneumoniae is a key virulence factor and typically conjugated with a carrier protein. It is necessary to improve the immunogenicity of the conjugate vaccine against S. pneumoniae. RESULTS: A phenyl linker between tetanus toxoid (TT) and S. pneumoniae Type 14 PS was used to improve the PS-specific immunogenicity of the conjugate vaccine. As compared with the one with the amyl linker (PS-TT), the conjugate with the phenyl linker (PS-phe-TT) decreased the TT-specific IgG titers and significantly increased the PS-specific IgG titers and the IL-5 level. CONCLUSION: The phenyl linker could potentiate a robust humoral immune response to PS by decreasing the carrier-induced epitopic suppression effect. PS-phe-TT was expected to act as an effective vaccine against S. pneumoniae.

Conjugation Reaction with 8-Arm PEG Markedly Improves the Immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 Fusion Protein.

Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen responsible for tuberculosis (TB). Effective vaccination is highly desired for immunoprotection against Mtb infection. CFP10 and TB10.4 are two important immunodominant Mtb-secreted protein antigens, which suffer from poor immunogenicity. Thus, an antigen delivery system and adjuvants are needed to improve the immunogenicity of the two proteins. A CFP10-TB10.4 fusion protein (CT) was used as the antigen in the present study. Conjugation of 4-6 CT molecules in one entity with 8-arm polyethylene glycol (PEG) acted as an antigen delivery system. Aluminum-loxoribine mixture (A-L) and poly(I:C) functioned as the adjuvants. As compared with CT, the polymerized CT (CT-PEG) elicited significantly higher CT-specific IgG titers, higher Th1- and Th2-type cytokines and higher percentages of CD4+ IFN-γ+ and CD4+ IL-4+ cells in BALB/c mice. The presence of A-L and poly(I:C) could both increase the immune response to CT-PEG. Conjugation reaction with 8-arm PEG showed a predominant driving force to improve the immunogenicity of CT. Pharmacokinetic study in SD rats revealed that conjugation reaction with 8-arm PEG prolonged the systemic circulation of CT and exposure to the immune system. CT-PEG with A-L showed no apparent toxicity to organs, whereas CT-PEG with poly(I:C) displayed some toxicity to organs. Thus, an effective and safe vaccine against Mtb infection could be rationally designed by conjugation reaction of Mtb-secreted protein antigen with 8-arm PEG and subsequent addition of A-L.

Conjugation of the CRM197-inulin conjugate significantly increases the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein.

Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen that causes tuberculosis (TB). Effective vaccination is urgently needed to deal with the serious threat from TB. Mtb-secreted protein antigens are important virulence determinants of Mtb with poor immunogenicity. Adjuvants and antigen delivery systems are thus highly desired to improve the immunogenicity of protein antigens. Inulin is a biocompatible polysaccharide (PS) adjuvant that can stimulate a strong cellular and humoral immunity. Bacterial capsular PS and haptens have been conjugated with cross-reacting material 197 (CRM197) to improve their immunogenicity. CFP10 and TB10.4 were two Mtb-secreted immunodominant protein antigens. A CFP10-TB10.4 fusion protein (CT) was used as the antigen for covalent conjugation with the CRM197-inulin conjugate (CRM-inu). The resultant conjugate (CT-CRM-inu) elicited high CT-specific IgG titers, stimulated splenocyte proliferation and provoked the secretion of Th1-type and Th2-type cytokines. Conjugation with CRM-inu significantly prolonged the systemic circulation of CT and exposure to the immune system. Moreover, CT-CRM-inu showed no apparent toxicity to cardiac, hepatic and renal functions. Thus, conjugation of CT with CRM-inu provided an effective strategy for development of protein-based vaccines against Mtb infection.

Potent Antigen-Adjuvant Delivery System by Conjugation of Mycobacterium tuberculosis Ag85B-HspX Fusion Protein with Arabinogalactan-Poly(I:C) Conjugate.

Protein-based vaccine is promising to improve or replace Mycobacterium bovis BCG vaccine for its specificity, safety, and easy production. However, protein-based vaccine calls for potent adjuvants and improved delivery systems to protect against Mycobacterium tuberculosis. Poly(I:C) is one of the most potent pathogen-associated molecular patterns that signals primarily via TLR3. Arabinogalactan (AG) is a biocompatible polysaccharide that can increase splenocyte proliferation and stimulate macrophages. The AG-poly(I:C) conjugate (AG-P) showed an adjuvant potency through a synergistic interaction of AG and poly(I:C). Ag85B and HspX are two important virulent protein antigens of Mycobacterium tuberculosis and Ag85B-HspX fusion protein (AH) was prepared. An antigen-adjuvant delivery system (AH-AG-P) was developed by conjugation of AH with AG-P to ensure that both AH and AG-P reach the APCs simultaneously. AH-AG-P elicited high AH-specific IgG titers and stimulated lymphocyte proliferation. AH-AG-P provoked the secretion of Th1-type cytokines (TNF-α, IFN-γ, and IL-2) and Th2-type cytokines (IL-4 and IL-10). Pharmacokinetics revealed that conjugation with AG-P could prolong the serum exposure of AH to the immune system. Pharmacodynamics suggested that conjugation with AG-P led to a rapid and intense production of AH-specific IgG. Accordingly, conjugation with AG-P could promote a robust cellular and humoral immune response to AH. Thus, conjugation of AH with a potent adjuvant AG-P is an effective strategy to develop an efficacious protein-based vaccine against Mycobacterium tuberculosis.

Conjugation with an Inulin-Chitosan Adjuvant Markedly Improves the Immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 Fusion Protein.

Protein-based vaccines are of potential to deal with the severe situations posed by Mycobacterium tuberculosis (Mtb). Due to inherently poor immunogenicity of Mtb protein antigens, a potent immunostimulatory adjuvant is needed to enhance the cellular and humoral immune response to Mtb protein antigens. Inulin and chitosan (Cs) are polysaccharide adjuvants that can be used to achieve such an objective. The inulin-Cs conjugate (inulin-Cs) acted as a potent adjuvant through a synergistic interaction of inulin and Cs. CFP10 and TB10.4 are two important virulent protein antigens of Mtb. The CFP10-TB10.4 fusion protein (CT) was constructed and used as the protein antigen. In the present study, an adjuvant delivery system (inulin-Cs-CT) was developed by covalent conjugation of CT with inulin-Cs. Conjugation with inulin-Cs significantly increased the hydrodynamic volume of CT and did not alter the structure of CT. High levels of Th1-type cytokines (IFN-γ, TNF-α, and IL-2) and Th2-type cytokine (IL-4) were secreted by provocation of inulin-Cs-CT. Inulin-Cs-CT elicited high CT-specific antibody titers, mostly in the form of IgG1 and IgG2b. Pharmacokinetics revealed that conjugation with inulin-Cs could prolong the serum exposure of CT to the immune system. Pharmacodynamics suggested that conjugation with inulin-Cs led to an efficient production of CT-specific IgG. Thus, conjugation of inulin-Cs can serve as a potent adjuvant delivery system to improve the immunogenicity of the Mtb protein antigens.