ABSTRACT
Influenza virus infections represent an ongoing public health threat as well as an economic burden. Although seasonal influenza vaccines have been available for some decades, efforts are being made to generate new efficient, flexible, and cost-effective technologies to be transferred into production. Our work describes the development of a model influenza hemagglutinin antigen that is capable of inducing protection against viral challenge in mice. High amounts of the H1 hemagglutinin ectodomain, HA18-528, were expressed in a bacterial system as insoluble inclusion bodies. Solubilization was followed by a thorough differential scanning fluorimetry (DSF)-guided optimization of refolding, which allows for fast and reliable screening of several refolding conditions, yielding tens of milligrams/L of folded protein. Structural and functional analysis revealed native-like folding as well as the presence of a mix of monomers and oligomers in solution. Mice immunized with HA18-528 were protected when exposed to influenza A virus as opposed to mice that received full-length denatured protein. Sera of mice immunized with HA18-528 showed both high titers of antigen-specific IgG1 and IgG2a isotypes as well as viral neutralization activity. These results prove the feasibility of the recombinant bacterial expression system coupled with DSF-guided refolding in providing influenza hemagglutinin for vaccine development.
ABSTRACT
Differential diagnosis of bacterial meningitis (BM) and viral meningitis (VM) is a critical clinical challenge, as the early and accurate identification of the causative agent determines the appropriate treatment regimen and markedly improves patient outcomes. Clinical and experimental studies have demonstrated that the pathogen and the host immune response contribute to mortality and neurological sequelae. As BM is associated with the activation of an inflammatory cascade, the patterns of pro- and anti-inflammatory cytokines/chemokines (CTs/CKs) present in the cerebrospinal fluid (CSF) in response to the immune assault may be useful as sensitive markers for differentiating BM from VM. In the present study, the ability of CTs/CKs in the CSF to differentiate between BM and VM was investigated. For this, biochemical markers and CT/CK profiles were analysed in 145 CSF samples, divided into three groups: BM (n=61), VM (n=58) and the control group (C; n=26) comprising patients with meningism. The CSF concentrations of monocyte chemoattractant protein-1, interleukin (IL)-8, IL-1ß, IL-6, macrophage inflammatory protein-1α (MIP-1α), epithelial-neutrophil activating peptide, IL-10, tumour necrosis factor-α (TNF-α), proteins and white blood cells were significantly higher and the CSF glucose level was significantly lower in the BM group compared with the VM and C groups (P<0.01). Correlation analysis identified 28 significant correlations between various CTs/CKs in the BM group (P<0.01), with the strongest positive correlations being for TNF-α/IL-6 (r=0.75), TNF-α/MIP-1α (r=0.69), TNF-α/IL-1ß (r=0.64) and IL-1ß/MIP-1α (r=0.64). To identify the optimum CT/CK patterns for predicting and classifying BM and VM, a dataset of 119 BM and VM samples was divided into training (n=90) and testing (n=29) subsets for use as input for a Random Forest (RF) machine learning algorithm. For the 29 test samples (15 BM and 14 VM), the RF algorithm correctly classified 28 samples, with 92% sensitivity and 93% specificity. The results show that the patterns of CT/CK levels in the CSF can be used to aid discrimination of BM and VM.
ABSTRACT
Protein immobilization using biopolymer scaffolds generally involves undesired protein loss of function due to denaturation, steric hindrance or improper orientation. Moreover, most methods for protein immobilization require expensive reagents and laborious procedures. This work presents the synthesis and proof of concept application of two alginate hydrogels that are able to bind proteins with polyhistidine tags by means of interaction with the crosslinking cations. Nickel (II) and cobalt (II) alginate hydrogels were prepared using a simple ionic gelation method. Hydrogels were characterized by optical microscopy and AFM, and evaluated for potential cytotoxicity. In addition, binding capacity was tested towards proteins with or without HisTAG. Hydrogels had moderate cytotoxicity and were able to exclusively bind polyhistidine-tagged proteins with a binding capacity of approximately 300 µg EGFP (enhanced green fluorescent protein) per 1 mL of hydrogel. A lyophilized hydrogel-protein complex dissolved upon the addition of PBS and allowed the protein release and regain of biological activity. In conclusion, the nickel (II) and cobalt (II) alginate biopolymers provided an excellent platform for the "carry and release" of polyhistidine-tagged proteins.
