Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh.

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.

Evaluation of the Safety, Tolerability and Immunogenicity of ShigETEC, an Oral Live Attenuated Shigella-ETEC Vaccine in Placebo-Controlled Randomized Phase 1 Trial.

BACKGROUND: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) cause high morbidity and mortality worldwide, yet no licensed vaccines are available to prevent corresponding infections. A live attenuated non-invasive Shigella vaccine strain lacking LPS O-antigen and expressing the ETEC toxoids, named ShigETEC was characterized previously in non-clinical studies. METHODS: ShigETEC was evaluated in a two-staged, randomized, double-blind and placebo-controlled Phase I clinical trial. A single dose of increasing amounts of the vaccine was given to determine the maximum tolerated dose and increasing number of immunizations were administered with an interval based on the duration of shedding observed. RESULTS: Oral immunization with ShigETEC was well tolerated and safe up to 4-time dosing with 5 × 1010 colony forming units. ShigETEC induced robust systemic immune responses against the Shigella vaccine strain, with IgA serum antibody dominance, as well as mucosal antibody responses evidenced by specific IgA in stool samples and in ALS (Antibodies in Lymphocyte Supernatant). Anti- ETEC toxin responses were detected primarily in the 4-times immunized cohort and for the heat-labile toxin correlated with neutralizing capacity. CONCLUSION: ShigETEC is a promising vaccine candidate that is scheduled for further testing in controlled human challenge studies for efficacy as well as in children in endemic setting for safety and immunogenicity.

Characterization of ShigETEC, a Novel Live Attenuated Combined Vaccine against Shigellae and ETEC.

Background: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) remain the two leading bacterial causes of diarrheal diseases worldwide. Attempts to develop preventive vaccines against Shigella and ETEC have not yet been successful. The major challenge for a broad Shigella vaccine is the serotype-specific immune response to the otherwise protective LPS O-antigen. ETEC vaccines mainly rely on the heat-labile enterotoxin (LT), while heat-stable toxin (ST) has also been shown to be an important virulence factor. Methods: We constructed a combined Shigella and ETEC vaccine (ShigETEC) based on a live attenuated Shigella strain rendered rough and non-invasive with heterologous expression of two ETEC antigens, LTB and a detoxified version of ST (STN12S). This new vaccine strain was characterized and tested for immunogenicity in relevant animal models. Results: Immunization with ShigETEC resulted in serotype independent protection in the mouse lung shigellosis model and induced high titer IgG and IgA antibodies against bacterial lysates, and anti-ETEC toxin antibodies with neutralizing capacity. Conclusions: ShigETEC is a promising oral vaccine candidate against Shigella and ETEC infections and currently in Phase 1 testing.

Application of FCS in studies of rhinovirus receptor binding and uncoating.

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.

Capillary electrophoresis, gas-phase electrophoretic mobility molecular analysis, and electron microscopy: effective tools for quality assessment and basic rhinovirus research.

We describe standard methods for propagation, purification, quality control, and physicochemical characterization of human rhinoviruses, using HRV-A2 as an example. Virus is propagated in HeLa-OHIO cells grown in suspension culture and purified via sucrose density gradient centrifugation. Purity and homogeneity of the preparations are assessed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), gas-phase electrophoretic mobility molecular analysis (GEMMA), and electron microscopy (EM). We also briefly describe usage of these methods for the characterization of subviral particles as well as for the analysis of their complexes with antibodies and soluble recombinant receptor mimics.

The Rhinovirus subviral a-particle exposes 3'-terminal sequences of its genomic RNA.

UNLABELLED: Enteroviruses, which represent a large genus within the family Picornaviridae, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (+)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that in vitro acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ∼700 bases. However, even after hours of incubation at pH 5.2, 5'-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment in vivo. This finding suggests that the exposed 3'-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded. IMPORTANCE: For host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification in vitro results in RNA exit starting from the 3'-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.

Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3'-end.

Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3'-end. This suggests that packaging also occurs in an ordered manner resulting in the 3'-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.