Biology subjects

Shaughnessy, J.

Publications and source records attributed to Shaughnessy, J..

4 recordsLinked to original sources

bioRxiv2026

Structural basis for recognition of a gonococcal lipooligosaccharide epitope by monoclonal antibody 2C7 informs vaccine and immunotherapeutic design

Neisseria gonorrhoeae causes gonorrhea and poses a growing global health threat driven by antimicrobial resistance and the lack of an effective vaccine. The lipooligosaccharide (LOS) epitope recognized by monoclonal antibody (mAb) 2C7 is expressed by most clinical isolates, making it an attractive therapeutic target. To determine the molecular basis for mAb 2C7 recognition, we conducted structural studies of mAb 2C7 in complex with an octasaccharide derived from LOS. NMR epitope mapping demonstrated that mAb 2C7 contacts two of three glycan chains: the {beta}-chain (lactose) and the {gamma}-chain (N-acetylglucosamine). The 1.6-[A] resolution crystal structure of the Fab 2C7-octasaccharide complex revealed that these chains make extensive hydrogen bonds and hydrophobic contacts within a cleft of the Fab. Molecular dynamics (MD) simulations showed that the {beta} and {gamma} chains adopt stable, specific conformations, contrasted with transient interactions of the chain. Informed by these structural data, we synthesized a tetrasaccharide comprising the {beta} and {gamma} chains joined by heptose II. The tetrasaccharide conjugated to a carrier protein bound mAb 2C7 by ELISA and Western blotting. Isothermal titration calorimetry showed that the tetrasaccharide and octasaccharide bound mAb 2C7 with similar affinities, indicating that the smaller oligosaccharide retains the key binding determinants. Binding was enthalpically driven, consistent with the polar interactions observed crystallographically and by MD. NMR experiments with the tetrasaccharide confirmed interactions with all four residues, establishing it as the minimal 2C7 epitope. Together, these studies provide a structural framework for rational design of glycan-based vaccines and antibody therapeutics against antimicrobial-resistant N. gonorrhoeae.

microbiology↗

bioRxiv2026

Pre-clinical efficacy of a C4BP hexameric IgG Fc fusion protein against Neisseria gonorrhoeae

Gonorrhea is the second most common bacterial sexually transmitted infection and affects about 80 million people worldwide annually. The causative agent, Neisseria gonorrhoeae, has become resistant to almost every antibiotic used for its treatment. There is no licensed vaccine against gonorrhea. Therefore, there is an urgent need to develop novel prevention and treatment strategies to curb the spread of gonorrhea. The gonococcus has evolved several mechanisms to evade complement, a key arm of immune defenses against this pathogen, including binding of the human complement inhibitors Factor H (FH) and C4b-binding protein (C4BP). We previously showed that chimeric molecules fusing the gonococcal binding domains of FH and C4BP to IgG Fc and IgM Fc, respectively, mediate complement-dependent killing of gonococci in vitro and attenuate gonococcal colonization of mouse vaginas when administered topically. Here, we fused C4BP domains 1 and 2, which contain the gonococcal binding region, to IgG Fc bearing the IgM tail-piece to facilitate Fc hexamerization. This molecule, called C4BP-Hexa IgG Fc, showed [~]650-fold greater complement-dependent bactericidal activity on a molar basis than monomeric C4BP-IgG1 Fc. C4BP-Hexa IgG Fc enhanced association with and uptake by human neutrophils in a complement-independent manner. Despite off-target complement activation in solution, C4BP-Hexa IgG Fc reduced both the duration and the bacterial burden of gonococcal vaginal colonization in human FH and C4BP transgenic mice when administered intravaginally daily. In conclusion, we show proof-of-concept of the efficacy of a hexameric C4BP IgG Fc fusion molecule against N. gonorrhoeae, which could aid in the fight against this multidrug-resistant pathogen.

immunology↗

bioRxiv2026

C4BP occludes the non-opsonic interaction of Neisseria gonorrhoeae with human neutrophil CEACAMs

Neisseria gonorrhoeae (Gc) causes the sexually transmitted infection gonorrhea, an urgent public health concern. Gc infection elicits a robust neutrophil response and serum leakage, but Gc has developed specialized defenses to evade both complement and neutrophils. We recently reported that the classical complement pathway inhibitor C4b-binding protein (C4BP) binds to Gc and reduces phagocytic killing by neutrophils in a complement-independent manner. Here, we used a Chinese hamster ovary (CHO) expression system and engineered C4BP constructs to define the underlying molecular mechanisms. C4BP inhibited interactions between opacity protein (Opa)-expressing Gc and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), receptors that drive non-opsonic phagocytosis of Gc by neutrophils. The degree of C4BP-mediated inhibition varied among CEACAMs. By using wild-type and chimeric CEACAMs, we found C4BP was more inhibitory towards the granulocyte-restricted CEACAM3 than the ubiquitously expressed CEACAM1, which we ascribed to CEACAM3s shorter extracellular domain. C4BP also inhibited the association between Opa-expressing Gc and the GPI-anchored CEACAM6. Molecules containing C4BP domains 1 and 2 fused to IgM (C4BP-IgM) or to a hexameric IgG Fc construct (C4BP-Hexa-IgG), proteins similar in diameter and degree of multimerization to native C4BP, inhibited the association of Opa-expressing Gc with CEACAM3-CHO cells to the same degree as C4BP, while C4BP domains 1 and 2 fused to dimeric Fc (C4BP-IgG) did not. C4BP-IgM, but not C4BP-IgG bearing mutations to abrogate Fc gamma receptor interactions, blocked Opa-mediated phagocytosis by primary human neutrophils. These results support a model in which C4BP occludes Opa-CEACAM interactions, which protects Gc from phagocytic killing by neutrophils.

microbiology↗

bioRxiv2025

The sialome of the retina, alteration in age-related macular degeneration (AMD) pathology and potential impacts on Complement Factor H

PurposeLittle is known about sialic acids of the human retina, despite their integral role in self/non-self-discrimination by complement factor H (CFH), the alternative complement pathway inhibitor. MethodsA custom sialoglycan microarray was used to characterize the sialic acid-binding specificity of native CFH or recombinant molecules where IgG Fc was fused to CFH domains 16-20 (contains a sialic acid-binding site), domains 6-7 (contains a glycosaminoglycan-binding site) or the CFH-related proteins (CFHRs) 1 and 3. We analyzed macular and peripheral retinal tissue from post-mortem ocular globes for amount, type, and presentation (glycosidic linkage type) of sialic acid in individuals with age-related macular degeneration (AMD) and age-matched controls using fluorescent lectins and antibodies to detect sialic acid and endogenous CFH. Released sialic acids from neural retina, retinal pigmented epithelium (RPE) cells and the Bruchs membrane (BrM) were labelled with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB), separated and quantified by high-performance liquid chromatography (DMB-HPLC). ResultsBoth native CFH and the recombinant CFH domains 16-20 recognized Neu5Ac and Neu5Gc that is 2-3-linked to the underlying galactose. 4-O-Actylation of sialic acid and sulfation of GlcNAc did not inhibit binding. Different linkage types of sialic acid were localized at different layers of the retina. The greatest density of 2-3-sialic acid, which is the preferred ligand of CFH, did not colocalize with endogenous CFH. The level of sialic acids at the BrM/choroid interface of macula and peripheral retina of individuals with AMD were significantly reduced. ConclusionsThe sialome of the human retina is altered in AMD. This can affect CFH binding and consequently, alternative complement pathway regulation.

biochemistry↗