Biology subjects

Gray-Owen, S.

Publications and source records attributed to Gray-Owen, S..

8 recordsLinked to original sources

bioRxiv2025

Structure-based engineering of a nutrient acquisition protein enhances neutralizing antibodies and protection for the development of a gonococcal vaccine

Gonorrhea is increasingly resistant to treatment and has been labelled an urgent threat due to the diminishing effectiveness of existing therapeutics. To address this challenge, we targeted the Neisseria gonorrhoeae transferrin binding protein B (TbpB), which is critical for iron acquisition and neisserial growth, as a vaccine target. Building on previous studies investigating the application of TbpB as an immunogen against various bacterial pathogens, we aimed to optimize this antigen for a broad protective effect. We compared the efficacy of wild type TbpB immunogens with engineered TbpB mutants that do not bind human transferrin (hTf) using infection studies in transgenic mice expressing hTf, which were required because the strict specificity of neisserial TbpB precludes its complexing with non-human transferrin. Comprehensive biophysical analyses confirmed that the introduced single residue mutations abolished hTf binding without compromising antigen structure. Immunization with the mutant antigens conferred increased resistance to infection by N. gonorrhoeae relative to that provided by the wild-type antigen in the humanized mice. When considering effector functions of the humoral response, we observed that the mutated antigen elicited more effective bactericidal and function-neutralizing activity. Through strategic mutations, we therefore enhanced vaccine effectiveness in a physiologically relevant model without significantly affecting the structure or immunogenicity of the antigen. This study highlights the use of rational structure-guided antigen design to drive effective immune responses and the potential interference of immunogen binding to host factors, and reinforces the utility of targeting TbpB in a gonococcal vaccine.

immunology↗

bioRxiv2025

Rationally designed minimized TbpB confers broad protection against meningococcal infection

Transferrin binding protein B (TbpB), an iron acquisition protein, has long been recognized as a promising vaccine candidate targeting the pathogenic Neisseria species, including Neisseria meningitidis, the cause of meningococcal disease, and Neisseria gonorrhoeae, the cause of gonorrhea. A challenge to the development of this protein as a vaccine immunogen is the extent of antigenic variability it exhibits, which complicates the selection of a single variant to elicit a broadly cross-protective immune response. We have utilized structure-informed antigen engineering to develop a minimized version of TbpB consisting of the proteins carboxy-terminal lobe with its variable surface loops removed. Here, we reveal the effectiveness of this "loopless C-lobe" as an independent immunogen, with structural characterization and stability studies to demonstrate its integrity, and murine immunization and challenge studies that establish its ability to elicit robust protective efficacy by using N. meningitidis invasive infection and nasopharyngeal colonization models. The breadth of protection provided, as measured by both in vitro analysis and cross-protection mouse challenge studies, indicate that a single loopless C-lobe elicits a broadly cross-protective immune response against the diverse panel of meningococcal strains tested, and that the cross-reactivity is superior to that offered by the intact TbpB or the native C-lobe. Together, this study demonstrates the utility of structure-informed antigen engineering towards the development of broadly efficacious protein-based vaccines. ImportanceSurface-exposed proteins on bacterial pathogens are enticing candidate vaccine targets, however their exposure to the immune system frequently leads to high levels of antigenic variation, a factor that complicates the development of broadly protective vaccines. Here, we undertake an antigen engineering approach to develop a minimized version of a surface lipoprotein, transferrin binding protein B, where variable regions of the protein have been removed to focus the immune response to conserved regions of this antigen. We combine structural studies and mouse infection models of Neisseria meningitidis, the cause of meningococcal disease, and Neisseria gonorrhoeae, the causative agent of gonorrhea, to reveal that our strategic minimizing of the protein immunogen focuses the immune response to extend the resulting breadth of cross-reactivity and cross-protection.

immunology↗

bioRxiv2025

Network-based population analysis of 31,595 gonococcal genomes reveals phase variability, genetic diversity and mobile element dynamics drive antimicrobial resistance and phenotypic diversity

With a highly plastic genome allowing the accumulation of antimicrobial resistance (AMR) determinants, Neisseria gonorrhoeae (Ngo) is an urgent public health threat. To better understand the spread of AMR in Ngo and its ability to persist in humans, we performed a systematic analysis of its global population structure. From 31,595 publicly available genomes, we curated a maximally diverse representative set of 485 gonococcal genomes, which were defined into 12 distinct clades on the basis of shared patterns of single nucleotide polymorphisms. Mapping AMR determinants and mobile elements onto these clades revealed significant correlation with gonococcal population structure. Notably, several variants associated with AMR were identified in genes not previously linked with resistance, suggesting roles as new determinants of resistance or compensatory mutations in resistant strains. Analysis of phase variable motifs, a significant mechanism driving regulation of expression in Ngo, identified 57 genes not previously identified as phase variable; in vivo and in vitro passaging validated our sequence-based definition of phase variability and revealed a strong association of phase variation of the surface proteins Opa and LgtG with improved colonization during infection. Together these findings highlight the ability of systematic comparative genomic analyses to shed new light on the drivers of Ngo population structure and identify new AMR determinants.

microbiology↗

bioRxiv2024

Phylogeny-based selection of representative variants with Navargator: Proof of principle using humoral cross-reactivity data from two immunization studies

BackgroundThe accessibility to the immune system of bacterial surface proteins makes them attractive targets for subunit vaccines. However, this same property also means they tend to exhibit high sequence variability. Achieving broad cross-protection usually necessitates that antigens from multiple isolates are included, but the choice of sequence variant is a non-trivial problem. Visual inspection of phylogenetic trees is the norm, but this is subjective and can be greatly influenced by the choice of viewing software. This has real-world implications, as groups have shown that the selection of non-optimal antigens likely led to lower cross-protection in the commercially available vaccines against Neisseria meningitidis serogroup B. Aim / MethodsTo address this problem, we have developed Navargator, bioinformatics software that takes a phylogenetic tree as input and identifies the variants that are the most similar to the greatest number of other sequences. The underlying premise is that cross-reactivity will be correlated with phylogenetic distances extracted from the tree; this was validated by several rodent immunization studies with the proteins transferrin-binding protein B and factor H binding protein from N. meningitidis and N. gonorrhoeae, measuring antibody-based cross-reactivity between an antigen panel using a custom high-throughput ELISA. ResultsNavargator has been made freely available both as an online tool and as source code for local installation. We implemented several different clustering methods, with exact algorithms for smaller datasets, and heuristics suitable for large trees of thousands of sequences. Our immunization studies have shown that this approach is sound, and that cross-reactivity is predicted well by phylogenetic distances in a sigmoidal manner. ConclusionsThe complexity of vaccine development rises sharply with each additional antigen included, so using the minimal number required is an important consideration. Navargator attempts to facilitate this in a systematic and generalizable manner. The user can run the analysis by selecting their desired number of representatives, or they can provide any form of cross-reactivity data and have the program identify a minimum reactivity threshold via correlation with the phylogenetic tree. The program will then identify the smallest number of representatives required to satisfy this threshold.

bioinformatics↗

bioRxiv2024

Protection against Neisseria meningitidis nasopharyngeal colonization relies on antibody opsonization and phagocytosis by neutrophils

Neisseria meningitidis is a human-restricted pathogen that can cause a rapidly progressing invasive meningococcal disease, yet it is also a regular inhabitant of the human nasopharynx. Vaccines that target N. meningitidis aim to prevent invasive disease, but their ability to interfere with nasal colonization could effectively eradicate this bacteria in a population, and so is an important target for meningococcal vaccine design. While protection against invasive meningococcal disease is classically attributed to IgG-dependent complement activation and bacterial killing, there remains no indication of what confers protection against nasopharyngeal colonization, making it impossible to deliberately target this stage during vaccine development. Moreover, without understanding what confers protection in this tissue site, it is impossible to understand the level of susceptibility within a population. To address this, we have taken advantage of the CEACAM1-humanized mouse model to characterize immune effectors that protect against nasal carriage of N. meningitidis. Protection against nasal colonization could be induced by live mucosal infection or by parenteral immunization with heat-killed bacteria. Mice possessing genetic deficiencies in B cells were used to evaluate the role of B cells and a specific antibody response, while neutrophil and complement depletion were used to evaluate their respective contributions to immunization-induced protection against meningococcal nasal carriage. Despite the essential role for complement killing in preventing invasive meningococcal disease, complement was not required for protection against nasal colonization. Instead, N. meningitidis-specific antibodies and neutrophils were both required to protect mice against the nasal infection. Combined, these data suggest that phagocytic bacterial killing is necessary for protection against mucosal colonization by N. meningitidis, indicating that nasal immunoglobulin with the ability to promote opsonophagocytosis must be considered as a correlate of protection against meningococcal carriage. AUTHORS SUMMARYNeisseria meningitidis can cause devastating and often fatal systemic infections including sepsis and meningitis, yet it frequently lives in the throat of healthy individuals. Vaccines developed against some meningococcal strains allow the individual to resist becoming colonized by the bacteria, an effect that protects them from disease and prevents them from spreading the bacteria to others, while other vaccines effectively protect against disease but still allow the individual to carry the bacteria in their throat. The reason for this difference has remained difficult to explain. Here, we use a humanized mouse model that allows N. meningitidis infection in the nasal passages to establish that effective protection against nasal colonization requires that antibodies present within the infected mucosal tissues can coat the bacteria so that they are engulfed by neutrophils, a potent bacteria-killing white blood cell that is recruited to the site of infection. These findings suggest that antibodies with the ability to promote neutrophil recognition and killing of N. meningitidis should be the goal of future vaccines, and the presence of these can be used to consider an individuals resistance against this terrible pathogen.

microbiology↗

bioRxiv2024

Human transferrin and lactoferrin cooperatively support Neisseria meningitidis colonization in the murine nasopharynx

Neisseria meningitidis is a human-restricted bacteria that is a normal nasopharyngeal resident, yet it can also disseminate, causing invasive meningococcal disease. Meningococci are highly adapted to life in humans, with human-specific virulence factors contributing to bacterial adhesion, nutrient acquisition and immune evasion. While these factors have been explored in isolation, their relative contribution during infection has not been considered due to their absence in small animal models and their expression by different human cell types not readily combined in either in vitro or ex vivo systems. Herein, we show that transgenic expression of the iron-binding glycoproteins human transferrin and lactoferrin can each facilitate N. meningitidis replication in mouse serum but that transferrin was required to support infection-induced sepsis. While these host proteins are insufficient to allow nasopharyngeal colonization alone, mice co-expressing these and human CEACAM1 support robust colonization. In this case, meningococcal colonization elicits an acute elevation in both transferrin and lactoferrin levels within the upper respiratory mucosa, with transferrin levels remaining elevated while lactoferrin returns to basal levels after establishment of infection. Competitive infection of triple transgenic animals with transferrin- and lactoferrin- binding protein mutants selects for bacteria expressing the transferrin receptor, implicating the critical contribution of transferrin-based iron acquisition to support colonization. These transgenic animals have thus allowed us to disentangle the relative contribution of three virulence factors during colonization and invasive disease, and provides a novel in vivo model that can support extended meningococcal colonization, opening a new avenue to explore the meningococcal lifestyle within its primary niche.

microbiology↗

bioRxiv2021

Correlated STORM-homoFRET Imaging: Applications to the Study of Membrane Receptor Self-Association and Clustering

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. We report here on a correlated STORM/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Live cell homoFRET imaging of CEACAM1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, revealed highly heterogenous diffraction-limited structures on the surface of HeLa cells. Correlated super-resolved STORM imaging revealed that these structures comprised a complex mixture and spatial distribution of self-associated CEACAM1 molecules. This correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

biophysics↗

bioRxiv2021

Intranasal HD-Ad Vaccine Protects the Upper and Lower Respiratory Tracts of hACE2 Mice against SARS-CoV-2

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

microbiology↗