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The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria., Competing Interests: Declaration of interests J.R.B., A.J.R.C., G.G., B.G.W., L.D.W.K., L.T.W., J.T., K. McHugh, and S.J.D. are inventors on patent applications relating to RH5 malaria vaccines and/or antibodies. A.M.M. and S.J.D. have consulted to GSK on malaria vaccines. A.M.M. has an immediate family member who is an inventor on patent applications relating to RH5 malaria vaccines and antibodies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin™ (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 μg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action., (Copyright 2010 Elsevier B.V. All rights reserved.)

This way up. Dual polarisation interferometry was used to design and characterise a surface on which the orientation and density of immobilised carbohydrates was suitable for studying their interactions with proteins. Lactoferrin was shown to adopt two orientations: "end-on" or "side-on", while for FGF-2 a single monolayer of protein was observed. The new surface can be used to elucidate the binding of proteins to carbohydrates and the geometry of the complexes, a frequently controversial area. Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index and mass of adsorbates 100 nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates is suitable for studying their interactions with proteins and where nonspecific binding is reduced to less than 5 % of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional crosslinker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulfate (KS) chondroitin sulfate (CS) and heparin, that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG. The heparin DP-10 surfaces were then used to determine the performance of the immobilised DP-10 with respect to binding of two well-characterised proteins, lactoferrin (Lf) and fibroblast growth factor-2. The results show that Lf could adopt two different orientations, at high protein loadings the protein layer thickness corresponded to an "end-on" orientation of Lf, whilst rinsing with buffer saw the Lf molecules adopt a "side-on" configuration. In the case of FGF-2, a single monolayer of protein bound to DP-10 was observed. These results demonstrate that the new surface can be used to resolve key questions relating to the binding of proteins to carbohydrates, including, when used in DPI, the resolution of the geometry of complexes, an area that is frequently controversial.

Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have investigated the dependence of the lytic activity of melittin on lipid composition. The lysis of liposomes, measured by following their mass and dimensions when immobilised on a solid substrate, was close to zero when the negatively charged lipids phosphatidyl glycerol or phosphatidyl serine were used as the phospholipid component of the liposome. Whilst there was significant binding of melittin to the liposomes, there was little net change in their diameter with melittin binding reversed upon salt injection. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain unsaturation, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state showed strong resistance to lysis. By directly measuring the dimensions and mass changes of liposomes on exposure to melittin using Dual Polarisation Interferometry, rather than following the florescence of entrapped dyes we attained further information about the initial stages of melittin binding to liposomes.

Dual polarisation interferometry (DPI) has been used to characterise the formation of hybrid bilayer membranes (HBM) on a silicon-oxynitride surface. This technique allows the simultaneous determination of multiple physical properties of an HBM, as the HBM is being formed in a single experiment: mass, thickness in the z-direction (normal to the surface), tilt angle of the first layer and refractive index. Decanoic acid was covalently attached to an amine modified silicon-oxynitride sensor chip surface via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride condensation reaction. The decanoic acid layer was 0.92+/-0.12 nm thick, indicating a tilt angle of 57 degrees from surface normal, and possessed a mass of 1.05+/-0.10 ng mm(-2) and a refractive index (RI) of 1.450+/-0.020. Phospholipid vesicles made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were injected over the fatty acid surface to form an HBM. The DPPC HBM was 4.32+/-0.68 nm thick, with a total mass of 3.18+/-0.60 ng mm(-2) and a RI of 1.404+/-0.007. The DMPC HBM was 2.12+/-0.34 nm thick, with a total mass of 2.25+/-0.51 ng mm(-2), and a RI of 1.435+/-0.007. DPI thus provides an insight into HBM formation and differences between the structural organisation of HBMs of different composition.

A new optical biosensor is described based on a dual waveguide interferometric technique. By addressing the waveguide structure with alternate polarisations the optogeometrical properties (density and thickness) of adsorbed protein layers at the sensor (solid)-liquid interface have been determined. Differences in the waveguide mode dispersion between the transverse electric (TE) and transverse magnetic (TM) modes allow unique solutions for adlayer thickness and refractive index to be determined at all stages during the formation process. The technique has been verified using standard protein systems and by comparing the data with published work using X-ray crystallography and neutron reflection techniques. The data obtained was found to be in excellent agreement with previously reported X-ray experiments given that typical film thicknesses for streptavidin layers were in the range 5.5-6.5 nm compared with the short axis crystal structure of between 4.8 and 5.6 nm. The precision of the measurements taken was of the order of 40 pm with respect to adsorbed adlayer thicknesses. This biosensor approach provides measurements of both thickness and density of adlayers to a high precision, simultaneously and in real time enabling detail of the structure and function of proteins to be elucidated. From such data it is possible to obtain information on the orientation, distortion and efficiency of immobilisation procedures as well as the interaction event of interest. The technique is expected to find utility with those interested in protein structure and function. This is an area of growing importance within the life sciences as the demand for quantitative analytical techniques increases with the growth in "proteomics".

Silicon is possibly important in human physiology in protecting against the toxic effects of aluminium, but the kinetics of uptake and excretion of silicic acid, the bioavailable form, are not well characterised. We have used 32Si as a tracer in a human uptake experiment to determine a gastrointestinal uptake factor for silicic acid, and to elucidate the kinetics of renal elimination. Urine collections were made for extending intervals from 2 to 12 h over 2 days following ingestion by a single human subject of a neutral silicic acid solution containing tracer levels of 32Si (t1/2 approximately 150 y). Silicon was isolated as SiO2 and the 32Si content determined by accelerator mass spectrometry (AMS), using a gas-filled magnet technique to eliminate a prolific isobaric interference from 32S. Silicon uptake appears to have been essentially complete within 2 h of ingestion. Elimination occurred by two simultaneous first-order processes with half-lives of 2.7 and 11.3 h, representing around 90% and 10%, respectively, of the total output. The rapidly eliminated 32Si was probably retained in the extracellular fluid volume, whilst the slower component may represent intracellular uptake and release. Elimination of absorbed 32Si was essentially complete after 48 h and was equivalent to 36% of the ingested dose. This establishes only a lower limit for gastrointestinal absorption as, although there was no evidence for longer term retention of additional 32Si, the possibility could not be excluded by these results.

Studies of the biological chemistry of aluminium can gain significantly from the use of the long-lived isotope 26Al as a tracer, although the cost of the isotope often precludes its determination by radiochemical counting techniques. Accelerator mass spectrometry (AMS) provides an ultra-sensitive method of determination, free from isobaric interference from atomic (26Mg) or molecular species. The source materials for AMS can be aluminium oxide or phosphate, both of which can be readily prepared at a sufficient level of purity from biological substrates. Natural aluminium (27Al, 100%) is added to the preparations as a chemical yield monitor and to provide the reference for the isotope ratio measurement. 26Al/27Al ratios can be determined over the range 10(-14)-10(-7), implying a limit of detection for 26Al of around 10(-18) g. The precision of measurement and long-term reproducibility are < 5% and < 7% (RSD), respectively. Chemical methodologies for routine measurements on blood and urine samples have been developed.