The Identification of Enteric Fever-Specific Antigens for Population-Based Serosurveillance.

BACKGROUND: Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low- and middle-income countries. Moderate sensitivity and scalability of current methods likely underestimate enteric fever burden. Determining the serological responses to organism-specific antigens may improve incidence measures. METHODS: Plasma samples were collected from blood culture-confirmed enteric fever patients, blood culture-negative febrile patients over the course of 3 months, and afebrile community controls. A panel of 17 Salmonella Typhi and Paratyphi A antigens was purified and used to determine antigen-specific antibody responses by indirect ELISAs. RESULTS: The antigen-specific longitudinal antibody responses were comparable between enteric fever patients, patients with blood culture-negative febrile controls, and afebrile community controls for most antigens. However, we found that IgG responses against STY1479 (YncE), STY1886 (CdtB), STY1498 (HlyE), and the serovar-specific O2 and O9 antigens were greatly elevated over a 3-month follow up period in S. Typhi/S. Paratyphi A patients compared to controls, suggesting seroconversion. CONCLUSIONS: We identified a set of antigens as good candidates to demonstrate enteric fever exposure. These targets can be used in combination to develop more sensitive and scalable approaches to enteric fever surveillance and generate invaluable epidemiological data for informing vaccine policies. CLINICAL TRIAL REGISTRATION: ISRCTN63006567.

An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic.

The epidemiology of typhoid fever in Lao People`s Democratic Republic is poorly defined. Estimating the burden of typhoid fever in endemic countries is complex due to the cost and limitations of population-based surveillance; serological approaches may be a more cost-effective alternative. ELISAs were performed on 937 serum samples (317 children and 620 adults) from across Lao PDR to measure IgG antibody titers against Vi polysaccharide and the experimental protein antigens, CdtB and HlyE. We measured the significance of the differences between antibody titers in adults and children and fitted models to assess the relationship between age and antibody titers. The median IgG titres of both anti-HylE and CdtB were significantly higher in children compared to adults (anti-HylE; 351.7 ELISA Units (EU) vs 198.1 EU, respectively; p<0.0001 and anti-CdtB; 52.6 vs 12.9 EU; p<0.0001). Conversely, the median anti-Vi IgG titer was significantly higher in adults than children (11.3 vs 3.0 U/ml; p<0.0001). A non-linear trend line fitted to the anti-CdtB and anti-HlyE IgG data identified a peak in antibody concentration in children <5 years of age. We identified elevated titers of anti-HlyE and anti-CdtB IgG in the serum of children residing in Lao PDR in comparison to adults. These antigens are associated with seroconversion after typhoid fever and may be a superior measure of disease burden than anti-Vi IgG. This approach is scalable and may be developed to assess the burden of typhoid fever in countries where the disease may be endemic, and evidence is required for the introduction of typhoid vaccines.

Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter.

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.

Stoichiometry and affinity of nucleotide binding to P-glycoprotein during the catalytic cycle.

Drug transport mediated by P-glycoprotein (Pgp) is driven by hydrolysis of ATP at the two cytosolic nucleotide binding domains. However, little is currently known concerning the stoichiometry of nucleotide binding and how both stoichiometry and binding affinity change during the catalytic cycle of the transporter. To address this issue, we used fluorescence techniques to measure both the number of nucleotides bound to P-glycoprotein during various stages of the catalytic cycle and the affinity of nucleotide binding. Results showed that resting state P-glycoprotein bound two molecules of the fluorescent nucleotide derivative, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), whereas the vanadate-trapped transition state bound only one nucleotide molecule. Both resting and transition state P-glycoprotein showed similar affinity for TNP-ATP/TNP-ADP and unlabeled ATP/ADP. Following binding of various drugs, resting state P-glycoprotein displayed a higher affinity for nucleotides, up to 4-fold depending on the compound used. In contrast, the transition state showed substantially lower (up to 3-fold) nucleotide binding affinity when the drug binding site(s) is/are occupied. These results indicate that both nucleotide binding domains of P-glycoprotein are likely to be occupied with either ATP (or ADP) in the resting state and the transition state in the absence of transport substrates. Drugs alter the binding affinity to favor association of ATP with P-glycoprotein at the start of the catalytic cycle and release of ADP from the transition state following nucleotide hydrolysis.