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Abstract Background CD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81). Findings Three-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop. Conclusions Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024. The Author(s).)

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

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., Competing Interests: VL, VP, and SM are employees of Sanofi-Pasteur. Part of the dengue VLP work was performed under an unrestricted research agreement between JT, University of Melbourne and Sanofi-Pasteur. There are two patents (PCT 35580344 and PCT 35580347) and three provisional patents (Patent Specification – 35541073, Patent Specification, SARS-CoV-2 VLP – 35555578, and Patent Specification, SARS-CoV-2 VLP – 35549058) covering this work. DG is a member of the Scientific Advisory Board of Avalia Immunotherapies. SG is a Director of Avalia Immunotherapies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Collett, Earnest, Carrera Montoya, Edeling, Yap, Wong, Christiansen, Roberts, Mumford, Lecouturier, Pavot, Marco, Loi, Simmons, Gulab, Mackenzie, Elbourne, Ramsland, Cameron, Hans, Godfrey and Torresi.)

The blood fluke Cardicola forsteri (Trematoda: Aporocotylidae) is a pathogen of ranched bluefin tuna in Japan and Australia. Genomics of Cardicola spp. have thus far been limited to molecular phylogenetics of select gene sequences. In this study, sequencing of the C. forsteri genome was performed using Illumina short-read and Oxford Nanopore long-read technologies. The sequences were assembled de novo using a hybrid of short and long reads, which produced a high-quality contig-level assembly (N50 > 430 kb and L50 = 138). The assembly was also relatively complete and unfragmented, comprising 66% and 7.2% complete and fragmented metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs), respectively. A large portion (> 55%) of the genome was made up of intergenic repetitive elements, primarily long interspersed nuclear elements (LINEs), while protein-coding regions cover > 6%. Gene prediction identified 8,564 hypothetical polypeptides, > 77% of which are homologous to published sequences of other species. The identification of select putative proteins, including cathepsins, calpains, tetraspanins, and glycosyltransferases is discussed. This is the first genome assembly of any aporocotylid, a major step toward understanding of the biology of this family of fish blood flukes and their interactions within hosts., Competing Interests: The authors have declared that no competing interests exist.

Helicobacter pylori is an important human pathogen that infects half the human population and can lead to significant clinical outcomes such as acute and chronic gastritis, duodenal ulcer, and gastric adenocarcinoma. To establish infection, H. pylori employs several mechanisms to overcome the innate and adaptive immune systems. H. pylori can modulate interleukin (IL) secretion and innate immune cell function by the action of several virulence factors such as VacA, CagA and the type IV secretion system. Additionally, H. pylori can modulate local dendritic cells (DC) negatively impacting the function of these cells, reducing the secretion of immune signaling molecules, and influencing the differentiation of CD4 + T helper cells causing a bias to Th1 type cells. Furthermore, the lipopolysaccharide (LPS) of H. pylori displays a high degree of phase variation and contains human blood group carbohydrate determinants such as the Lewis system antigens, which are proposed to be involved in molecular mimicry of the host. Lastly, the H. pylori group of outer membrane proteins such as BabA play an important role in attachment and interaction with host Lewis and other carbohydrate antigens. This review examines the various mechanisms that H. pylori utilises to evade the innate immune system as well as discussing how the structure of the H. pylori LPS plays a role in immune evasion., Competing Interests: Author AG was employed by company ZiP Diagnostics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sijmons, Guy, Walduck and Ramsland.)

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., (Copyright © 2021 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Finding an effective therapeutic to prevent or cure AD has been difficult due to the complexity of the brain and limited experimental models. This study utilized unmodified and genetically modified Saccharomyces cerevisiae as model organisms to find potential natural bioactive compounds capable of reducing intracellular amyloid beta 42 (Aβ 42 ) and associated oxidative damage. Eleven natural bioactive compounds including mangiferin, quercetin, rutin, resveratrol, epigallocatechin gallate (EGCG), urolithin A, oleuropein, rosmarinic acid, salvianolic acid B, baicalein and trans -chalcone were screened for their ability to reduce intracellular green fluorescent protein tagged Aβ 42 (GFP-Aβ 42 ) levels. The two most effective compounds from the screens were combined in varying concentrations of each to study the combined capacity to reduce GFP-Aβ 42 . The most effective combinations were examined for their effect on growth rate, turnover of native Aβ 42 and reactive oxygen species (ROS). The bioactive compounds except mangiferin and urolithin A significantly reduced intracellular GFP-Aβ 42 levels. Baicalein and trans -chalcone were the most effective compounds among those that were screened. The combination of baicalein and trans -chalcone synergistically reduced GFP-Aβ 42 levels. A combination of 15 μM trans -chalcone and 8 μM baicalein was found to be the most synergistic combination. The combination of the two compounds significantly reduced ROS and Aβ 42 levels in yeast cells expressing native Aβ 42 without affecting growth of the cells. These findings suggest that the combination of baicalein and trans -chalcone could be a promising multifactorial therapeutic strategy to cure or prevent AD. However, further studies are recommended to look for similar cytoprotective activity in humans and to find an optimal dosage.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T-cell reactivity., Methods: We investigated the differences between four tropomyosins-the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)-in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model., Results: Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (T m 42°C) and Der p 10 (T m 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (T m 63°C) or Ani s 3 (T m 33°C). Pen m 1-specific T-cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T-cell epitope cross-reactivity., Conclusions: Tropomyosin T-cell cross-reactivity, unlike IgE cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross-sensitization among different invertebrates and design of suitable T-cell peptide-based immunotherapies for shrimp and related allergies., (© 2020 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Because of the ongoing increase in antibiotic-resistant microbes, new strategies such as therapeutic antibodies and effective vaccines are required. Bacterial carbohydrates are known to be particularly antigenic, and several monoclonal antibodies that target bacterial polysaccharides have been generated, with more in current development. This review examines the known 3D crystal structures of anti-bacterial antibodies and the structural basis for carbohydrate recognition and explores the potential mechanisms for antibody-dependent bacterial cell death. Understanding the key interactions between an antibody and its polysaccharide target on the surface of bacteria or in biofilms can provide essential information for the development of more specific and effective antibody therapeutics as well as carbohydrate-based vaccines., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Glycans are complex sugar chains, crucial to many biological processes. By participating in binding interactions with proteins, glycans often play key roles in host-pathogen interactions. The specificities of glycan-binding proteins, such as lectins and antibodies, are governed by motifs within larger glycan structures, and improved characterisations of these determinants would aid research into human diseases. Identification of motifs has previously been approached as a frequent subtree mining problem, and we extend these approaches with a glycan notation that allows recognition of terminal motifs., Results: In this work, we customised a frequent subtree mining approach by altering the glycan notation to include information on terminal connections. This allows specific identification of terminal residues as potential motifs, better capturing the complexity of glycan-binding interactions. We achieved this by including additional nodes in a graph representation of the glycan structure to indicate the presence or absence of a linkage at particular backbone carbon positions. Combining this frequent subtree mining approach with a state-of-the-art feature selection algorithm termed minimum-redundancy, maximum-relevance (mRMR), we have generated a classification pipeline that is trained on data from a glycan microarray. When applied to a set of commonly used lectins, the identified motifs were consistent with known binding determinants. Furthermore, logistic regression classifiers trained using these motifs performed well across most lectins examined, with a median AUC value of 0.89., Conclusions: We present here a new subtree mining approach for the classification of glycan binding and identification of potential binding motifs. The Carbohydrate Classification Accounting for Restricted Linkages (CCARL) method will assist in the interpretation of glycan microarray experiments and will aid in the discovery of novel binding motifs for further experimental characterisation.

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5-2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy., (© 2020 Soliman et al.)

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6-24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 μg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin., Competing Interests: The authors have declared that no competing interests exist.

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In order to facilitate a number of processes including nutrient acquisition and immune evasion, malaria parasites extensively remodel their host erythrocyte. This remodelling is to a large extent accomplished through protein export, a crucial process mediated by the Plasmodium translocon for exported proteins (PTEX) translocon which is comprised of three core components, HSP101, PTEX150 and EXP2. EXP2 has been structurally and electrophysiologically shown to form the pore that spans the vacuole membrane enveloping the parasite. Here, we biochemically investigate the structure and function of EXP2. By differential alkylation we provide direct evidence that cysteines C113 and C140 form an intramolecular disulphide bond, while C201 is predominantly in a reduced state. We demonstrate that EXP2 possesses a protease resistant, membrane-associated, N-terminal region of ∼20 kDa that does not project into the infected erythrocyte cytosol; however, its C-terminus does project into the vacuole space. We show that a putative transmembrane peptide derived from the N-terminal region of EXP2 is haemolytic and in a polymer-based osmotic protection assay, we demonstrate that this peptide forms a discrete haemolytic pore. This work provides further biochemical insight into the role, function and cellular arrangement of EXP2 as the pore-forming component for protein translocation., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Summary: A sliding window analysis over a protein or genomic sequence is commonly performed, and we present a Python tool, BioStructMap, that extends this concept to three-dimensional (3D) space, allowing the application of a 3D sliding window analysis over a protein structure. BioStructMap is easily extensible, allowing the user to apply custom functions to spatially aggregated data. BioStructMap also allows mapping of underlying genomic sequences to protein structures, allowing the user to perform genetic-based analysis over spatially linked codons-this has applications when selection pressures arise at the level of protein structure., Availability and Implementation: The Python BioStructMap package is available at https://github.com/andrewguy/biostructmap and released under the MIT License. An online server implementing standard functionality is available at https://biostructmap.burnet.edu.au., Supplementary Information: Supplementary data are available at Bioinformatics online.

Bacterial adhesion to human epithelia via lectins constitutes a therapeutic opportunity to prevent infection. Specifically, BambL (the lectin from Burkholderia ambifaria) is implicated in cystic fibrosis, where lectin-mediated bacterial adhesion to fucosylated lung epithelia is suspected to play an important role. We employed structure-based virtual screening to identify inhibitors of BambL-saccharide interaction with potential therapeutic value. To enable such discovery, a virtual screening protocol was iteratively developed via 194 retrospective screening protocols against 4 bacterial lectins (BambL, BC2L-A, FimH, and LecA) with known ligands. Specific attention was given to the rigorous evaluation of retrospective screening, including calculation of analytical errors for enrichment metrics. The developed virtual screening workflow used crystallographic constraints, pharmacophore filters, and a final manual selection step. The protocol was applied to BambL, predicting 15 active compounds from virtual libraries of approximately 7 million compounds. Experimental validation using fluorescence polarization confirmed micromolar inhibitory activity for two compounds, which were further characterized by isothermal titration calorimetry and surface plasmon resonance. Subsequent testing against LecB from Pseudomonas aeruginosa demonstrated binding specificity of one of the hit compounds. This report demonstrates the utility of virtual screening protocols, integrating ligand-based pharmacophore filtering and structure-based constraints, in the search for bacterial lectin inhibitors.

Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Le y ) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Le y -positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Le y on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Plasmodium vivax is a significant contributor to the global malaria burden, and a vaccine targeting vivax malaria is urgently needed. An understanding of the targets of functional immune responses during the course of natural infection will aid in the development of a vaccine. Antibodies play a key role in this process, with responses against particular epitopes leading to immune selection pressure on these epitopes. A number of techniques exist to estimate levels of immune selection pressure on particular epitopes, with a sliding window analysis often used to determine particular regions likely to be under immune pressure. However, such analysis neglects protein three-dimensional structural information. With this in mind, a newly developed tool, BioStructMap, was applied to two key antigens from Plasmodium vivax: PvAMA1 and PvDBP Region II. This tool incorporates structural information into tests of selection pressure., Results: Sequences from a number of populations were analysed, examining spatially-derived nucleotide diversity and Tajima's D over protein structures for PvAMA1 and PvDBP. Structural patterns of nucleotide diversity were similar across all populations examined, with Domain I of PvAMA1 having the highest nucleotide diversity and displaying significant signatures of immune selection pressure (Tajima's D > 0). Nucleotide diversity for PvDBP was highest bordering the dimerization and DARC-binding interface, although there was less evidence of immune selection pressure on PvDBP compared with PvAMA1. This study supports previous work that has identified Domain I as the main target of immune-mediated selection pressure for PvAMA1, and also supports studies that have identified functional epitopes within PvDBP Region II., Conclusions: The BioStructMap tool was applied to leading vaccine candidates from P. vivax, to examine structural patterns of selection and diversity across a number of geographic populations. There were striking similarities in structural patterns of diversity across multiple populations. Furthermore, whilst regions of high diversity tended to surround conserved binding interfaces, a number of protein regions with very low diversity were also identified, and these may be useful targets for further vaccine development, given previous evidence of functional antibody responses against these regions.

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly- N -acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N -acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Humoral immune responses against the malaria parasite are an important component of a protective immune response. Antibodies are often directed towards conformational epitopes, and the native structure of the antigenic region is usually critical for antibody recognition. We examined the structural features of various Plasmodium antigens that may impact on epitope location, by performing a comprehensive analysis of known and modelled structures from P. falciparum. Examining the location of known polymorphisms over all available structures, we observed a strong propensity for polymorphic residues to be exposed on the surface and to occur in particular secondary structure segments such as hydrogen-bonded turns. We also utilised established prediction algorithms for B-cell epitopes and MHC class II binding peptides, examining predicted epitopes in relation to known polymorphic sites within structured regions. Finally, we used the available structures to examine polymorphic hotspots and Tajima's D values using a spatial averaging approach. We identified a region of PfAMA1 involving both domains II and III under a high degree of balancing selection relative to the rest of the protein. In summary, we developed general methods for examining how sequence-based features relate to one another in three-dimensional space and applied these methods to key P. falciparum antigens.

Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in respiratory infection. X-ray crystallography has revealed the bound complex structures for four fucosylated human blood group epitopes (blood group B, H type 1, H type 2, and Le x determinants). The present study employed computational approaches, including docking and molecular dynamics (MD), to extend the structural analysis of BambL-oligosaccharide complexes to include four additional blood group saccharides (A, Le a , Le b , and Le y ) and a library of blood-group-related carbohydrates. Carbohydrate recognition is dominated by interactions with fucose via a hydrogen-bonding network involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36, and Trp74. BambL recognition is dominated by interactions with fucose, but also features interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site provides guidelines for the future design of lectin inhibitors.

Carbohydrate-binding antibodies and carbohydrate-based vaccines are being actively pursued as targeted immunotherapies for a broad range of cancers. Recognition of tumor-associated carbohydrates (glycans) by antibodies is predominantly towards terminal epitopes on glycoproteins and glycolipids on the surface of cancer cells. Crystallography along with complementary experimental and computational methods have been extensively used to dissect antibody recognition of glycan epitopes commonly found in cancer. We provide an overview of the structural biology of antibody recognition of tumor-associated glycans and propose potential rearrangements of these targets in the membrane that could dictate the complex biological activities of these antibodies against cancer cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n = 12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells in vivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers., (© 2017 John Wiley & Sons Ltd.)

Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies., Summary: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 10 12 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s -1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs., (© 2017 International Society on Thrombosis and Haemostasis.)

Background: The aim of the study was to explore Fc mutations of a humanised anti-Lewis-Y antibody (IgG1) hu3S193 as a strategy to improve therapeutic ratios for therapeutic payload delivery., Methods: Four hu3S193 variants (I253A, H310A, H435A and I253A/H310A) were generated via site-directed mutagenesis and radiolabelled with diagnostic isotopes iodine-125 or indium-111. Biodistribution studies in Lewis-Y-positive tumour-bearing mice were used to calculate the dose in tumours and organs for therapeutic isotopes (iodine-131, yttrium-90 and lutetium-177)., Results: (111)In-labelled I253A and H435A showed similar slow kinetics (t 1/2β, 63.2 and 62.2 h, respectively) and a maximum tumour uptake of 33.11 ± 4.05 and 33.69 ± 3.77 percentage injected dose per gramme (%ID/g), respectively. (111)In-labelled I253A/H310A cleared fastest (t 1/2β, 9.1 h) with the lowest maximum tumour uptake (23.72 ± 0.85 %ID/g). The highest increase in tumour-to-blood area under the curve (AUC) ratio was observed with the metal-labelled mutants ((90)Y and (177)Lu). (177)Lu-CHX-A" DTPA-hu3S193 I253A/H310A (6:1) showed the highest tumour-to-blood AUC ratio compared to wild type (3:1) and other variants and doubling of calculated dose to tumour based on red marrow dose constraints., Conclusions: These results suggest that hu3S193 Fc can be engineered with improved therapeutic ratios for (90)Y- and (177)Lu-based therapy, with the best candidate being hu3S193 I253A/H310A for (177)Lu-based therapy.

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc-protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc-binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc-protein complexes are available, numerous others have not yet been determined. Protein-protein docking could be used to investigate Fc-protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking-based structural bioinformatics approach is developed for predicting the structures of Fc-protein complexes. Based on the available set of X-ray structures of Fc-protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc-protein complexes, as well as in the design of peptides and proteins that target Fc., (Copyright © 2016 John Wiley & Sons, Ltd.)

We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.

Malaria remains a significant global health burden. The development of an effective malaria vaccine remains as a major challenge with the potential to significantly reduce morbidity and mortality. While Plasmodium spp. have been shown to contain a large number of intrinsically disordered proteins (IDPs) or disordered protein regions, the relationship of protein structure to subcellular localisation and adaptive immune responses remains unclear. In this study, we employed several computational prediction algorithms to identify IDPs at the proteome level of six Plasmodium spp. and to investigate the potential impact of protein disorder on adaptive immunity against P. falciparum parasites. IDPs were shown to be particularly enriched within nuclear proteins, apical proteins, exported proteins and proteins localised to the parasitophorous vacuole. Furthermore, several leading vaccine candidates, and proteins with known roles in host-cell invasion, have extensive regions of disorder. Presentation of peptides by MHC molecules plays an important role in adaptive immune responses, and we show that IDP regions are predicted to contain relatively few MHC class I and II binding peptides owing to inherent differences in amino acid composition compared to structured domains. In contrast, linear B-cell epitopes were predicted to be enriched in IDPs. Tandem repeat regions and non-synonymous single nucleotide polymorphisms were found to be strongly associated with regions of disorder. In summary, immune responses against IDPs appear to have characteristics distinct from those against structured protein domains, with increased antibody recognition of linear epitopes but some constraints for MHC presentation and issues of polymorphisms. These findings have major implications for vaccine design, and understanding immunity to malaria.

Background: The targets and mechanisms of human immunity to malaria are poorly understood, which poses a major barrier to malaria vaccine development. Antibodies play a key role in human immunity and may act by inhibiting receptor-binding functions of key merozoite invasion ligands. Antibodies to the major invasion ligand and vaccine candidate, erythrocyte-binding antigen 175 (EBA-175), have been linked with protection, but how these antibodies function has not been established., Methods: We developed 2 new assays that quantify the ability of antibodies to inhibit binding of EBA-175 to its erythrocyte receptor, glycophorin A, using either native or recombinant EBA-175. Binding-inhibitory antibodies were evaluated in a longitudinal cohort study of Papua New Guinean children and related to risk of malaria, age, infection status, and markers of parasite exposure., Results: Binding-inhibition assays (BIAs) were reproducible, and the 2 assays had a high level of agreement. Inhibitory antibodies were common among children, acquired in association with markers of increasing parasite exposure, and high in those children with active infection. Inhibitory antibodies correlated with total immunoglobulin G levels to the EBA-175 binding domain (region II). Importantly, binding-inhibitory antibodies were significantly associated with protection from symptomatic malaria when measured using either BIA., Conclusions: Findings suggest that naturally acquired binding-inhibitory antibodies are an important functional mechanism that contributes to protection against malaria and further supports the potential of EBA-175 as a vaccine candidate. Identifying vaccines and approaches that induce potent binding-inhibitory antibodies may be a valuable strategy in the development of highly efficacious malaria vaccines., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Monoclonal antibodies represent the most successful class of biopharmaceuticals for the treatment of cancer. Mechanisms of action of therapeutic antibodies are very diverse and reflect their ability to engage in antibody-dependent effector mechanisms, internalize to deliver cytotoxic payloads, and display direct effects on cells by lysis or by modulating the biological pathways of their target antigens. Importantly, one of the universal changes in cancer is glycosylation and carbohydrate-binding antibodies can be produced to selectively recognize tumor cells over normal tissues. A promising group of cell surface antibody targets consists of carbohydrates presented as glycolipids or glycoproteins. In this review, we outline the basic principles of antibody-based targeting of carbohydrate antigens in cancer. We also present a detailed structural view of antibody recognition and the conformational properties of a series of related tissue-blood group (Lewis) carbohydrates that are being pursued as potential targets of cancer immunotherapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Molecular docking is a computational method for predicting the placement of ligands in the binding sites of their receptor(s). In this review, we discuss the methodological developments that occurred in the docking field in 2012 and 2013, with a particular focus on the more difficult aspects of this computational discipline. The main challenges and therefore focal points for developments in docking, covered in this review, are receptor flexibility, solvation, scoring, and virtual screening. We specifically deal with such aspects of molecular docking and its applications as selection criteria for constructing receptor ensembles, target dependence of scoring functions, integration of higher-level theory into scoring, implicit and explicit handling of solvation in the binding process, and comparison and evaluation of docking and scoring methods., (Copyright © 2015 John Wiley & Sons, Ltd.)

Monoclonal antibodies are being developed as therapeutics to complement drugs and vaccines or to fill the gap where no drugs or vaccines exist. These therapeutic antibodies (ThAb) may be especially important for infectious diseases in which there is antibiotic resistance, toxin-mediated pathogenesis, or for emerging pathogens. The unique structure of antibodies determines the specific nature of the effector function, so when developing ThAb, the desired effector functions need to be considered and integrated into the design and development processes to ensure maximum efficacy and safety. Antibody subclass is a critical consideration, but it is noteworthy that almost all ThAb that are licenced or currently in development utilise an IgG1 backbone. This review outlines the major structural properties that vary across subclasses, how these properties affect functional immunity, and discusses the various approaches used to study subclass responses to infectious diseases. We also review the factors associated with the selection of antibody subclasses when designing ThAb and highlight circumstances where different subclass properties might be beneficial when applied to particular infectious diseases. These approaches are critical to the future design of ThAb and to the study of naturally-acquired and vaccine-induced immunity., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lactic acid and short chain fatty acids (SCFAs) produced by vaginal microbiota have reported antimicrobial and immune modulatory activities indicating their potential as biomarkers of disease and/or disease susceptibility. In asymptomatic women of reproductive-age the vaginal microbiota is comprised of lactic acid-producing bacteria that are primarily responsible for the production of lactic acid present at ~110 mM and acidifying the vaginal milieu to pH ~3.5. In contrast, bacterial vaginosis (BV), a dysbiosis of the vaginal microbiota, is characterized by decreased lactic acid-producing microbiota and increased diverse anaerobic bacteria accompanied by an elevated pH>4.5. BV is also characterized by a dramatic loss of lactic acid and greater concentrations of mixed SCFAs including acetate, propionate, butyrate, and succinate. Notably women with lactic acid-producing microbiota have more favorable reproductive and sexual health outcomes compared to women with BV. Regarding the latter, BV is associated with increased susceptibility to sexually transmitted infections (STIs) including HIV. In vitro studies demonstrate that lactic acid produced by vaginal microbiota has microbicidal and virucidal activities that may protect against STIs and endogenous opportunistic bacteria as well as immune modulatory properties that require further characterization with regard to their effects on the vaginal mucosa. In contrast, BV-associated SCFAs have far less antimicrobial activity with the potential to contribute to a pro-inflammatory vaginal environment. Here we review the composition of lactic acid and SCFAs in respective states of eubiosis (non-BV) or dysbiosis (BV), their effects on susceptibility to bacterial/viral STIs and whether they have inherent microbicidal/virucidal and immune modulatory properties. We also explore their potential as biomarkers for the presence and/or increased susceptibility to STIs.

MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.