Characterization and predictive functional profiles on metagenomic 16S rRNA data of liver transplant recipients: A longitudinal study.

Long-term survival after Liver Transplantation (LT) is often compromised by infectious and metabolic complications. We aimed to delineate alterations in intestinal microbiome (IM) over time that could contribute to medical complications compromising long-term survival following LT. Fecal samples from LT recipients were collected at 3 months (3 M) and 6 months (6 M) post-LT. The bacterial DNA was extracted using E.Z.N.A. Stool DNA Kit and 16S rRNA gene sequencing at V4 hypervariable region was performed. DADA2 and Phyloseq was implemented to analyze the taxonomic composition. Differentially abundant taxa were identified by metagenomeSeq and LEfSe. Piphillin, an Inferred functional metagenomic analysis tool was used to study the bacterial functional content. For comparison, healthy samples were extracted from NCBI and analyzed similarly. The taxonomic & functional profiles of LT recipients were validated with metagenomic sequencing data from animals exposed to immunosuppressants using Venny. Our findings provide a new perspective on longitudinal increase in specific IM communities post-LT along with an increase in bacterial genes associated with metabolic and infectious disease.

Adjuvant Potential of Poly-α-l-Glutamine from the Cell Wall of Mycobacterium tuberculosis.

Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-α-l-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.

Characterization of a two component system, Bas1213-1214, important for oxidative stress in Bacillus anthracis.

Microbial colonization is an outcome of appropriate sensing and regulation of its gene expression. Bacillus anthracis adapts and thrives in its environment through complex regulatory mechanisms, among them, the two component systems (TCS). Many bacteria respond to the oxygen fluctuations via TCS. In the present work, a previously uncharacterized TCS, Bas1213-1214, of B. anthracis with a probable role in oxygen sensing has been characterized as a functional TCS. A substantial increase in the expression of Bas1213 was observed during the stationary growth phase, in presence of bicarbonate ions, and under oxidative stress thereby speculating the role of Bas1213 in toxin production and adaptive responses. Electrophoretic mobility shift assay (EMSA) and ANS assay highlighted autoregulation of the system. Identification of Bas1213 regulon further suggested its regulatory function in metabolism and adaptive responses. A marked reduction in sporulation was observed on overexpression of Bas1213 in B. anthracis which can be correlated with the augmented expression of sporulation kinase D. Additionally, Bas1213 was shown to regulate catalase, and ABC transporter (mntH) further implicating its essential role during oxidative stress. Finally, crucial residues involved in the DNA binding activity of Bas1213 were also identified. This study reports that the role of Bas1213-1214 in the regulation of metabolism and adaptive responses during oxidative stress. Both sporulation and response to environmental oxygen are important for the maintenance of B. anthracis lifecycle, therefore, characterization of Bas1213-1214 provides a step closer toward understanding the regulatory network governing in B. anthracis.

Erratum: Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo: Erratum.

[This corrects the article DOI: 10.7150/ntno.54879.].

Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo.

Extracellular vesicles (EVs) are naturally released, cell-derived vesicles that mediate intracellular communication, in part, by transferring genetic information and, thus, have the potential to be modified for use as a therapeutic gene or drug delivery vehicle. Advances in EV engineering suggest that directed delivery can be accomplished via surface alterations. Here we assess enriched delivery of engineered EVs displaying an organ targeting peptide specific to the pancreas. We first characterized the size, morphology, and surface markers of engineered EVs that were decorated with a recombinant protein specific to pancreatic ß-cells. This ß-cell-specific recombinant protein consists of the peptide p88 fused to the EV-binding domain of lactadherin (C1C2). These engineered EVs, p88-EVs, specifically bound to pancreatic ß-cells in culture and transferred encapsulated plasmid DNA (pDNA) as early as in 10 min suggesting that the internalization of peptide-bearing EVs is a rapid process. Biodistribution of p88-EVs administrated intravenously into mice showed an altered pattern of EV localization and improved DNA delivery to the pancreas relative to control EVs, as well as an accumulation of targeting EVs to the pancreas using luciferase activity as a readout. These findings demonstrate that systemic administration of engineered EVs can efficiently deliver their cargo as gene carriers to targeted organs in live animals.

Co-Administration of Aluminium Hydroxide Nanoparticles and Protective Antigen Domain 4 Encapsulated Non-Ionic Surfactant Vesicles Show Enhanced Immune Response and Superior Protection against Anthrax.

Aluminium salts have been the adjuvant of choice in more than 100 licensed vaccines. Here, we have studied the synergistic effect of aluminium hydroxide nanoparticles (AH np) and non-ionic surfactant-based vesicles (NISV) in modulating the immune response against protective antigen domain 4 (D4) of Bacillus anthracis. NISV was prepared from Span 60 and cholesterol, while AH np was prepared from aluminium chloride and sodium hydroxide. AH np was co-administered with NISV encapsulating D4 (NISV-D4) to formulate AHnp/NISV-D4. The antigen-specific immune response of AHnp/NISV-D4 was compared with that of commercial alhydrogel (alhy) co-administered with NISV-D4 (alhydrogel/NISV-D4), NISV-D4, AHnp/D4, and alhydrogel/D4. Co-administration of NISV-D4 with AH np greatly improved the D4-specific antibody titer as compared to the control groups. Based on IgG isotyping and ex vivo cytokine analysis, AHnp/NISV-D4 generated a balanced Th1/Th2 response. Furthermore, AH np/NISV-D4 showed superior protection against anthrax spore challenge in comparison to other groups. Thus, we demonstrate the possibility of developing a novel combinatorial nanoformulation capable of augmenting both humoral and cellular response, paving the way for adjuvant research.

Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax.

INTRODUCTION: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. METHODS AND RESULTS: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. CONCLUSION: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

Single-dose Ag85B-ESAT6-loaded poly(lactic-co-glycolic acid) nanoparticles confer protective immunity against tuberculosis.

BACKGROUND: Bacillus Calmette-Guérin, the attenuated strain of Mycobacterium bovis, remains the only available vaccine against tuberculosis (TB). However, its ineffectiveness in adults against pulmonary TB and varied protective efficacy (0-80%) speak to an urgent need for the development of an improved and efficient TB vaccine. In this milieu, poly(lactic-co-glycolic acid) (PLGA), is a preferential candidate, due to such properties as biocompatibility, targeted delivery, sustained antigen release, and atoxic by-products. METHODS: In this study, we formulated PLGA nanoparticles (NPs) encapsulating the bivalent H1 antigen, a fusion of Mycobacterium tuberculosis (Mtb) Ag85B and ESAT6 proteins, and investigated its role in immunomodulation and protection against Mtb challenge. Using the classical water-oil-water solvent-evaporation method, H1-NPs were prepared, with encapsulation efficiency of 86.1%±3.2%. These spherical NPs were ~244.4±32.6 nm in diameter, with a negatively charged surface (ζ-potential -4±0.6 mV). RESULTS: Under physiological conditions, NPs degraded slowly and the encapsulated H1 antigen was released over a period of weeks. As a proof-of-concept vaccine candidate, H1 NPs were efficiently internalized by the THP-1 human macrophages. Six weeks after a single-dose vaccination, H1 NP-immunized C57BL/6J mice showed significant increase in the production of total serum IgG (P<0.0001) and its isotypes compared to H1 alone, IgG2a being the predominant one, followed by IgG1. Further, the cytokine-release profile of antigen-stimulated splenocyteculture supernatant indicated a strong TH1-biased immunoresponse in H1 NP-vaccinated mice, with ~6.03- and ~2.8-fold increase in IFNγ and TNFα cytokine levels, and ~twofold and 1.6 fold increase in IL4 and IL10 cytokines, respectively, compared to H1 alone-immunized mice. In protection studies, H1 NP-vaccinated mice displayed significant reductions in lung and spleen bacillary load (P<0.05) at 5-week post-Mtb H37Rv challenge and prolonged survival, with a mean survival time of 177 days, compared to H1 alone-vaccinated mice (mean survival time 80 days). CONCLUSION: Altogether, our findings highlight the significance of the H1-PLGA nanoformulation in terms of providing long-term protection in mice with a single dose.

In-vitro Detection of Phytopathogenic Fungal Cell Wall by Polyclonal Sera Raised Against Trimethyl Chitosan Nanoparticles.

PURPOSE: The objective of this research was to generate a tool for the first-line detection of fungal infection in plants. Chitin is one of the unique fungal cell wall polysaccharide which is naturally deacetylated to chitosan upon infection. It is said to be involved in the fungal cell wall modulation and plant-pathogen communication. Therefore, detection of chitosan could be potentially helpful in the detection of fungal contamination. METHODS: Five different phytopathogenic fungi strains were used for the study. Polyclonal sera were raised in the mice against Trimethylchitosan nanoparticles to generate an enhanced humoral immune response and generate a rich and heterogeneous repertoire of antibodies. The binding affinity of the sera with fungal cell wall was analyzed by ELISA, Langmuir isotherm, confocal microscopy and ITC (Isothermal Calorimetry). RESULTS: The raised polyclonal sera could detect chitosan in the fungal cell wall, as analyzed with the different techniques. However, the detection specificity varied among the strains in proportion to the chitin content of their cell wall. Fusarium oxysporum was detected with the highest affinity while Trichoderma reesei was detected with the least affinity by ELISA. Adsorption isotherm, as well as ITC, revealed the specific and high binding capacity. Confocal microscopy also confirmed the detection of all strains used in the study. CONCLUSION: This novel technique employing TMC nanoparticulate system could be potentially used as a source to raise sera against chitosan in an inexpensive and less laborious manner. Rapid detection of fungal contamination by the polyclonal antibodies could help in devising a quick solution. The polyclonal sera are expected to detect a span of epitopes and provide precise detection. The detection system could be advanced for future applications such as food quality control, crop protection, and human fungal infection detection and treatment.

Novel application of trimethyl chitosan as an adjuvant in vaccine delivery.

The application of natural carbohydrate polysaccharides for antigen delivery and its adjuvanation potential has garnered interest in the scientific community in the recent years. These biomaterials are considered favorable candidates for adjuvant development due to their desirable properties like enormous bioavailability, non-toxicity, biodegradability, stability, affordability, and immunostimulating ability. Chitosan is the one such extensively studied natural polymer which has been appreciated for its excellent applications in pharmaceuticals. Trimethyl chitosan (TMC), a derivative of chitosan, possesses these properties. In addition it has the properties of high aqueous solubility, high charge density, mucoadhesive, permeation enhancing (ability to cross tight junction), and stability over a range of ionic conditions which makes the spectrum of its applicability much broader. It has also been seen to perform analogously to alum, complete Freund's adjuvant, incomplete Freund's adjuvant, and cyclic guanosine monophosphate adjuvanation, which justifies its role as a potent adjuvant. Although many review articles detailing the applications of chitosan in vaccine delivery are available, a comprehensive review of the applications of TMC as an adjuvant is not available to date. This article provides a comprehensive overview of structural and chemical properties of TMC which affect its adjuvant characteristics; the efficacy of various delivery routes for TMC antigen combination; and the recent advances in the elucidation of its mechanism of action.

Trimethyl Chitosan Nanoparticles Encapsulated Protective Antigen Protects the Mice Against Anthrax.

Anthrax is an era old deadly disease against which there are only two currently available licensed vaccines named anthrax vaccine adsorbed and precipitated (AVP). Though they can provide a protective immunity, their multiple side-effects owing to their ill-defined composition and presence of toxic proteins (LF and EF) of Bacillus anthracis, the causative organism of anthrax, in the vaccine formulation makes their widespread use objectionable. Hence, an anthrax vaccine that contains well-defined and controlled components would be highly desirable. In this context, we have evaluated the potential of various vaccine formulations comprising of protective antigen (PA) encapsulated trimethyl-chitosan nanoparticles (TMC-PA) in conjunction with either CpG-C ODN 2395 (CpG) or Poly I:C. Each formulation was administered via three different routes, viz., subcutaneous (SC), intramuscular (IM), and intraperitoneal in female BALB/c mice. Irrespective of the route of immunization, CpG or Poly I:C adjuvanted TMC-PA nanoparticles induced a significantly higher humoral response (total serum IgG and its isotypes viz., IgG1, IgG2a, and IgG2b), compared to their CpG or Poly I:C PA counterparts. This clearly demonstrates the synergistic behavior of CpG and Poly I:C with TMC nanoparticles. The adjuvant potential of TMC nanoparticles could be observed in all the three routes as the TMC-PA nanoparticles by themselves induced IgG titers (1-1.5 × 105) significantly higher than both CpG PA and Poly I:C PA groups (2-8 × 104). The effect of formulations on T-helper (Th) cell development was assessed by quantifying the Th1-dependant (TNF-α, IFN-γ, and IL-2), Th2-dependant (IL-4, IL-6, and IL-10), and Th17-type (IL-17A) cytokines. Adjuvanation with CpG and Poly I:C, the TMC-PA nanoparticles triggered a Th1 skewed immune response, as suggested by an increase in the levels of total IgG2a along with IFN-γ cytokine production. Interestingly, the TMC-PA group showed a Th2-biased immune response. Upon challenge with the B. anthracis Ames strain, CpG and Poly I:C adjuvanted TMC-PA nanoparticles immunized via the SC and IM routes showed the highest protective efficacy of ~83%. Altogether, the results suggest that CpG or Poly I:C adjuvanted, PA-loaded TMC nanoparticles could be used as an effective, non-toxic, second generation subunit-vaccine candidate against anthrax.