Probing the expression and adhesion of glycans involved in Helicobacter pylori infection.

Helicobacter pylori infects approximately half the human population and has an unusual infective niche of the human stomach. Helicobacter pylori is a major cause of gastritis and has been classified as a group 1 carcinogen by the WHO. Treatment involves triple or quadruple antibiotic therapy, but antibiotic resistance is becoming increasingly prevalent. Helicobacter pylori expresses certain blood group related antigens (Lewis system) as a part of its lipopolysaccharide (LPS), which is thought to assist in immune evasion. Additionally, H. pylori LPS participates in adhesion to host cells alongside several adhesion proteins. This study profiled the carbohydrates of H. pylori reference strains (SS1 and 26695) using monoclonal antibodies (mAbs) and lectins, identifying interactions between two carbohydrate-targeting mAbs and multiple lectins. Atomic force microscopy (AFM) scans were used to probe lectin and antibody interactions with the bacterial surfaces. The selected mAb and lectins displayed an increased adhesive force over the surface of the curved H. pylori rods. Furthermore, this study demonstrates the ability of anti-carbohydrate antibodies to reduce the adhesion of H. pylori 26695 to human gastric adenocarcinoma cells via AFM. Targeting bacterial carbohydrates to disrupt crucial adhesion and immune evasion mechanisms represents a promising strategy for combating H. pylori infection.

Adenovirus vector produced Zika virus-like particles induce a long-lived neutralising antibody response in mice.

Countermeasures against Zika virus (ZIKV) epidemics are urgently needed. In this study we generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. The ZIKV-VLPs were morphologically similar to ZIKV by electron microscopy and were recognized by anti-Flavivirus neutralising antibodies. We observed that a single dose of unadjuvanted ZIKV-VLPs, or inactivated ZIKV, generated an immune response that lasted over 6 months, but did not neutralize ZIKV infection of cells in vitro. However, when we co-administered the ZIKV VLPs with either Aluminium hydroxide (Alhydrogel®; Alum), AddaVax or Pam2Cys we observed that Alum was the most effective in a single dose regime, since it not only produced antibodies that neutralized the virus, but also generated a greater number of antigen-specific memory B cells. We additionally observed that the generation of the neutralising antibodies persisted for up to 6 months. Our results suggest that a single dose ZIKV VLPs could be a suitable single dose vaccine candidate for use in outbreak settings.

Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates.

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.

Profiling the glycome of Cardicola forsteri, a blood fluke parasitic to bluefin tuna.

Infections by blood flukes (Cardicola spp.) are considered the most significant health issue for ranched bluefin tuna, a major aquaculture industry in Japan and Australia. The host-parasite interfaces of trematodes, namely their teguments, are particularly rich in carbohydrates, which function both in evasion and modulation of the host immune system, while some are primary antigenic targets. In this study, histochemistry and mass spectrometry techniques were used to profile the glycans of Cardicola forsteri. Fluorescent lectin staining of adult flukes indicates the presence of oligomannose (Concanavalin A-reactive) and fucosylated (Pisum sativum agglutinin-reactive) N-glycans. Additionally, reactivity of succinylated wheat germ agglutinin (s-WGA) was localised to several internal organs of the digestive and monoecious reproductive systems. Glycan structures were further investigated with tandem mass spectrometry, which revealed structures indicated by lectin reactivity. While O-glycans from these adult specimens were not detectable by mass spectrometry, several oligomannose, paucimannosidic, and complex-type N-glycans were identified, including some carrying hexuronic acid and many carrying core xylose. This is, to our knowledge, the first glycomic characterisation of a marine platyhelminth, with broader implications for research into other trematodes.

Investigating virus-host cell interactions: Comparative binding forces between hepatitis C virus-like particles and host cell receptors in 2D and 3D cell culture models.

Cell cultures have been successfully used to study hepatitis C virus (HCV) for many years. However, most work has been done using traditional, 2-dimensional (2D) cell cultures (cells grown as a monolayer in growth flasks or dishes). Studies have shown that when cells are grown suspended in an extra-cellular-matrix-like material, they develop into spherical, 'organoid' arrangements of cells (3D growth) that display distinct differences in morphological and functional characteristics compared to 2D cell cultures. In liver organoids, one key difference is the development of clearly differentiated apical and basolateral surfaces separated and maintained by cellular tight junctions. This phenomenon, termed polarity, is vital to normal barrier function of hepatocytes in vivo. It has also been shown that viruses, and virus-like particles, interact very differently with cells derived from 2D as compared to 3D cell cultures, bringing into question the usefulness of 2D cell cultures to study virus-host cell interactions. Here, we investigate differences in cellular architecture as a function of cell culture system, using confocal scanning laser microscopy, and determine differences in binding interactions between HCV virus-like particles (VLPs) and their cognate receptors in the different cell culture systems using atomic force microscopy (AFM). We generated organoid cultures that were polarized, as determined by localization of key apical and basolateral markers. We found that, while uptake of HCV VLPs by both 2D and 3D Huh7 cells was observed by flow cytometry, binding interactions between HCV VLPs and cells were measurable by AFM only on polarized cells. The work presented here adds to the growing body of research suggesting that polarized cell systems are more suitable for the study of HCV infection and dynamics than non-polarized systems.

Probing and pressing surfaces of hepatitis C virus-like particles.

Hepatitis C virus-like particles (VLPs) are being developed as a quadrivalent vaccine candidate, eliciting both humoral and cellular immune responses in animal trials. Biophysical, biomechanical and biochemical properties are important for virus and VLP interactions with host cells and recognition by the immune system. Atomic force microscopy (AFM) is a powerful tool for visualizing surface topographies of cells, bionanoparticles and biomolecules, and for determining biophysical and biomechanical attributes such as size and elasticity. In this work, AFM was used to define morphological and nanomechanical properties of VLPs representing four common genotypes of hepatitis C virus. Significant differences in size of the VLPs were observed, and particles demonstrated a wide range of elasticity. Ordered packing of the core and potentially envelope glycoproteins was observed on the surfaces of the VLPs, but detailed structural characterization was hindered due to intrinsic dynamic fluctuations or AFM probe-induced damage of the VLPs. All VLPs were shown to be glycosylated in a manner similar to native viral particles. Together, the results presented in this study further our understanding of the nanostructure of hepatitis C VLPs, and should influence their uptake as viable vaccine candidates.

Imaging the air-water interface: Characterising biomimetic and natural hydrophobic surfaces using in situ atomic force microscopy.

The interface between water and a textured hydrophobic surface can exist in two regimes; either the Wenzel (surface-engulfed) or Cassie-Baxter (water-suspended) state. Better understanding of the influence of pattern geometry and spacing is crucial for the design of functional (super)hydrophobic surfaces, as inspired by numerous examples in nature. In this work, we have employed amplitude modulated - atomic force microscopy to visualize the air-water interface with an unprecedented degree of clarity on a superhydrophobic and a highly hydrophobic nanostructured surface. The images obtained provide the first real-time experimental visualization of the Cassie-Baxter wetting on the surface of biomimetic silicon nanopillars and a naturally superhydrophobic cicada wing. For both surfaces, the air-water interface was found to be remarkably well-defined, revealing a distinctly nanostructured air-water interface in the interstitial spacing. The degree of interfacial texture differed as a function of surface geometry. These results reveal that the air-water interface is heterogeneous in its structure and confirmed the presence of short-range interfacial ordering. Additionally, the overpressure values for each point on the interface were calculated, quantifying the difference in wetting behavior for the biomimetic and natural surface. Results suggest that highly-ordered, closely spaced nanofeatures facilitate robust Cassie-Baxter wetting states and therefore, can enhance the stability of (super)hydrophobic surfaces.

Rapid quantification of methamphetamine: using attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) and chemometrics.

In Australia and increasingly worldwide, methamphetamine is one of the most commonly seized drugs analysed by forensic chemists. The current well-established GC/MS methods used to identify and quantify methamphetamine are lengthy, expensive processes, but often rapid analysis is requested by undercover police leading to an interest in developing this new analytical technique. Ninety six illicit drug seizures containing methamphetamine (0.1%-78.6%) were analysed using Fourier Transform Infrared Spectroscopy with an Attenuated Total Reflectance attachment and Chemometrics. Two Partial Least Squares models were developed, one using the principal Infrared Spectroscopy peaks of methamphetamine and the other a Hierarchical Partial Least Squares model. Both of these models were refined to choose the variables that were most closely associated with the methamphetamine % vector. Both of the models were excellent, with the principal peaks in the Partial Least Squares model having Root Mean Square Error of Prediction 3.8, R(2) 0.9779 and lower limit of quantification 7% methamphetamine. The Hierarchical Partial Least Squares model had lower limit of quantification 0.3% methamphetamine, Root Mean Square Error of Prediction 5.2 and R(2) 0.9637. Such models offer rapid and effective methods for screening illicit drug samples to determine the percentage of methamphetamine they contain.

High-throughput optimization of surfaces for antibody immobilization using metal complexes.

Using a high-throughput surface discovery approach, we have generated a 1600-member library of metal-containing surfaces and screened them for antibody binding potential. The surface library assembly involved graft modification of argon plasma-treated polyvinylidenedifluoride (PVDF) membranes with alternating maleic anhydride-styrene copolymer followed by anhydride ring opening with a range of secondary amines and microarray contact printing of transition metal complexes. The microarrays of metal-containing surfaces were then tested for their antibody binding capacity by incubation with a biotinylated mouse antibody in a chemiluminescence assay. A total of 11 leads were identified from the first screen, constituting a "hit" rate of 0.7%. A smaller 135-member surface library was then synthesized and screened to optimize existing hits and generate additional leads. To demonstrate the applicability of these surfaces to other formats, high-binding surface leads were then transferred onto Luminex beads for use in a bead flow cytometric immunoassay. The novel one-step antibody coupling process increased assay sensitivity of a Luminex tumor necrosis factor immunoassay. These high-binding surfaces do not require prior incorporation of polyhistidine tags or posttreatments such as oxidation to achieve essentially irreversible binding of immunoglobulin G.