ABSTRACT
A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F-specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Nanoparticles/chemistry , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/chemistry , Viral Fusion Proteins/immunology , Animals , Antibody Formation , Antigens, Viral/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Respiratory Syncytial Virus Vaccines/chemistry , Respiratory Syncytial Virus, Human/immunology , Viral Fusion Proteins/chemistryABSTRACT
Cholera toxin (CT), the principal virulence factor secreted by Vibrio cholerae, is an A-B5 type exotoxin that binds to host cell GM1-gangliosides and is responsible for cholera diarrhoea. We tested the hypothesis that the cyclic hexasaccharide α-cyclodextrin (α-CD), but not the cyclic heptasaccharides methyl-ß-cyclodextrin (MD-ß-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD) inhibit binding of CT to GM1-gangliosides. We report that α-CD decreases CT binding to GM1-ganglioside-coated microtitre plate wells and on the surface of fixed HeLa cells in a concentration-dependent manner, suggesting that this may be a promising lead for the development of compounds with therapeutic properties.
Subject(s)
Cholera Toxin/chemistry , Gangliosides/chemistry , alpha-Cyclodextrins/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin , Enzyme-Linked Immunosorbent Assay , HeLa Cells , Humans , alpha-Cyclodextrins/chemistry , beta-Cyclodextrins/chemistryABSTRACT
Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis , are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1-10 µM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells and, surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA's cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins.
Subject(s)
Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Protein Multimerization/drug effects , Proteolysis/drug effects , Small Molecule Libraries/pharmacology , Animals , Antigens, Bacterial/toxicity , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/toxicity , Cell Line , Computational Biology , Drug Evaluation, Preclinical , Humans , Mice , Molecular Docking Simulation , Porosity , Protein Structure, Quaternary , Small Molecule Libraries/metabolismABSTRACT
Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.