Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea, is emerging as a critical health concern because of its rapidly escalating antibiotic resistance. The World Health Organization (WHO) has included Ng in the Bacterial Priority Pathogen List, primarily due to its increasing diffusion and resistance to first line treatments (fluoroquinolones and third-generation cephalosporins), thus raising the specter of untreatable infections. In the absence of new antibiotics, alternative tools are needed to fight the spreading of gonococcal infection, like monoclonal antibodies and vaccines. Among these, two complementary research directions are being investigated: (i) the development of a vaccine to specifically prevent gonococcal infection and (ii) the investigation of cross-protection between Neisseria gonorrhoeae and Neisseria meningitidis. A key focus in the field of gonococcal vaccine development is the use of outer membrane vesicles (OMVs) technology. OMVs are small, spherical, bilayered vesicles shed by gram negative bacteria during growth (or, alternatively, extracted from bacterial pellet with a detergent). OMVs maintain a native-like composition of bacterial surface antigens, including lipooligosaccharide (LOS) and proteins. OMVs expose both classes of antigens at the same time, thus can be used to study how their relative abundance, structure and spatial arrangement can impact immune recognition. In fact, LOS side chains are often longer and more flexible than proteins, they can potentially mask protein epitopes, affecting antibody generation and/or binding. To study the interplay between LOS and protein antigens, OMVs generated by N. gonorrhoeae expressing different LOS phenotypes were analyzed via two structural mass spectrometry techniques: Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) and Crosslinking Mass Spectrometry (XL-MS). Porin B (PorB), the most abundant outer membrane protein in N. gonorrhoeae and a potential vaccine candidate, was selected as a protein of interest. HDX-MS data revealed that LOS influences the exposure of PorB loop 1. In OMVs where LOS is absent, in fact, loop 1 is observed to be more accessible, which allows the immune system to generate antibodies targeting this region. Conversely, anti-loop 1 antibodies are not able to bind their epitope in presence of LOS, supporting the masking role of LOS towards neighboring protein antigens. These findings were in line with XL-MS results, where different crosslinking patterns were observed for the loop 1 of PorB when embedded in OMV with different LOS phenotypes. In parallel, epidemiological evidence has shown that vaccination with OMV-based vaccines against N. meningitidis serogroup B (MenB) is associated with a reduced incidence of gonococcal infection, despite these vaccines being originally designed to target meningococci. This observation has triggered various studies aimed at identifying the molecular basis of this shared antigenicity between the two species. For the experiment presented in this manuscript, sera from MenB vaccinees were screened to isolate monoclonal antibodies (mAbs) capable of binding gonococcus. Most of these antibodies were directed either against LOS or PorB, which was identified as a shared and immunogenic target between N. meningitidis and N. gonorrhoeae. Among anti-PorB antibodies, mAb 01K12 showed the highest bactericidal activity against gonococcus. Thanks to HDX-MS, the epitope recognized by mAb 01K12 was mapped on both meningococcal and gonococcal PorB embedded in detergent extracted outer membrane vesicles (dOMVs). In summary, this work highlights the complex interplay between saccharide and protein antigens in OMV-based vaccines and reveals how LOS structural variation can influence protein antigens accessibility to the immune system and antibody generation. These findings contribute to the knowledge to support the rational design of next-generation vaccines and immunotherapeutics for multidrug-resistant N. gonorrhoeae.
Neisseria gonorrhoeae (Ng), l'agente eziologico della gonorrea, è un preoccupante batterio dal punto di vista sanitario a causa della sua rapida e crescente resistenza agli antibiotici. A causa della sua crescente diffusione e resistenza ai trattamenti di prima linea (fluorochinoloni e cefalosporine di terza generazione), l'organizzazione mondiale della sanità (OMS) lo ha inserito tra gli high priority pathogens. In assenza di nuovi antibiotici, è necessario trovare strumenti alternativi per combattere la diffusione di questa infezione, come ad esempio anticorpi monoclonali e vaccini. Riguardo questi ultimi, due direzioni di ricerca sono attualmente in fase di studio: (i) lo sviluppo di un vaccino specifico per prevenire l'infezione di Ng (ii) l'indagine sulla cross-protezione tra Neisseria gonorrhoeae e Neisseria meningitidis. Le Outer Membrane Vesicles (OMVs) sono piccole vescicole di forma sferica, composte da un doppio strato fosfolipidico e generate dai batteri gram negativi durante la crescita (o, alternativamente, estratte dal pellet batterico con l'aiuto di detergenti). Le OMVs possono essere utilizzate come vaccini, mantengono una composizione simile a quella degli antigeni che si trovano naturalmente sulla superficie batterica, inclusi lipooligosaccaride (LOS) e proteine. Poichè entrambe le classi di antigeni sono esposte contemporaneamente, le OMVs si possono utilizzare per studiare l'abbondanza relativa, la struttura e l'organizzazione spaziale degli antigeni possono influenzare il sistema immunitario. Le catene laterali dei saccaridi, infatti, sono spesso più lunghe e flessibili delle proteine e possono potenzialmente mascherare gli epitopi proteici, influenzando così la generazione e/o il legame degli anticorpi. In questa tesi, l'interazione tra LOS e antigeni proteici è stata studiata analizzando OMVs generate da N. gonorrhoeae esprimenti diversi fenotipi di LOS tramite due tecniche di spettrometria di massa strutturale: (i) Spettrometria di massa con scambio idrogeno/deuterio (HDX-MS) (ii) Crosslinking mass spectrometry (HDX-MS) La Porina B (PorB), la più abbondante proteina di membrana in N. gonorrhoeae, è un potenziale candidato vaccino ed è stato selezionato come antigene modello in questa tesi. I dati ottenuti tramite HDX-MS rivelano che il LOS influenza l'esposizione del Loop 1 di PorB. Nelle OMVs private del LOS, infatti, il Loop 1 è più accessibile e il sistema immunitario è in grado di generare anticorpi specifici per questa regione. Gli anticorpi anti-Loop1 non riescono a legarsi al loro epitopo in presenza di LOS, confermando che i saccaridi possono mascherare gli antigeni proteici adiacenti. Anche gli esperimenti di crosslinking mostrano diversi pattern di reattività per il loop 1 per la PorB contenuta in vescicole esprimenti diversi fenotipi di LOS. Parallelamente, diverse evidenze epidemiologiche hanno mostrato che la vaccinazione contro N. meningitidis del sierogruppo B (MenB) è associata a una ridotta incidenza dell'infezione gonococcica. In seguito a questa osservazione, ci sono stati diversi studi per identificare gli antigeni responsabili di questo fenomeno. In questo lavoro, i sieri di individui vaccinati con Bexsero, un vaccino per la prevenzione dell'infezione meningococcica, sono stati analizzati per isolare gli anticorpi monoclonali (mAbs) in grado di legarsi a gonococco. La maggior parte di questi anticorpi era diretta contro il LOS o PorB, antigeni identificati come condivisi e immunogenici tra N. meningitidis e N. gonorrhoeae. Tra gli anticorpi anti-PorB, il mAb 01K12 ha mostrato la più alta attività battericida contro gonococco. Grazie alla spettrometria di massa HDX, l'epitopo riconosciuto da 01K12 è stato mappato sia sulla PorB di meningococco che di gonococco, entrambi inseriti in OMV estratte con un detergente (dOMVs). In sintesi, questo studio evidenzia la complessa interazione tra antigeni saccaridici e proteici nei vaccini OMV-based e rivela come la variazione strutturale del LOS possa influenzare l'accessibilità degli antigeni proteici al sistema immunitario e la generazione di anticorpi. Questi risultati supportano il design di vaccini e immunoterapie di nuova generazione contro N. gonorrhoeae multiresistente.
Role of lipooligosaccharide on conformation and accessibility of gram-negative antigens
FAVARON, SARA
2024/2025
Abstract
Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea, is emerging as a critical health concern because of its rapidly escalating antibiotic resistance. The World Health Organization (WHO) has included Ng in the Bacterial Priority Pathogen List, primarily due to its increasing diffusion and resistance to first line treatments (fluoroquinolones and third-generation cephalosporins), thus raising the specter of untreatable infections. In the absence of new antibiotics, alternative tools are needed to fight the spreading of gonococcal infection, like monoclonal antibodies and vaccines. Among these, two complementary research directions are being investigated: (i) the development of a vaccine to specifically prevent gonococcal infection and (ii) the investigation of cross-protection between Neisseria gonorrhoeae and Neisseria meningitidis. A key focus in the field of gonococcal vaccine development is the use of outer membrane vesicles (OMVs) technology. OMVs are small, spherical, bilayered vesicles shed by gram negative bacteria during growth (or, alternatively, extracted from bacterial pellet with a detergent). OMVs maintain a native-like composition of bacterial surface antigens, including lipooligosaccharide (LOS) and proteins. OMVs expose both classes of antigens at the same time, thus can be used to study how their relative abundance, structure and spatial arrangement can impact immune recognition. In fact, LOS side chains are often longer and more flexible than proteins, they can potentially mask protein epitopes, affecting antibody generation and/or binding. To study the interplay between LOS and protein antigens, OMVs generated by N. gonorrhoeae expressing different LOS phenotypes were analyzed via two structural mass spectrometry techniques: Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) and Crosslinking Mass Spectrometry (XL-MS). Porin B (PorB), the most abundant outer membrane protein in N. gonorrhoeae and a potential vaccine candidate, was selected as a protein of interest. HDX-MS data revealed that LOS influences the exposure of PorB loop 1. In OMVs where LOS is absent, in fact, loop 1 is observed to be more accessible, which allows the immune system to generate antibodies targeting this region. Conversely, anti-loop 1 antibodies are not able to bind their epitope in presence of LOS, supporting the masking role of LOS towards neighboring protein antigens. These findings were in line with XL-MS results, where different crosslinking patterns were observed for the loop 1 of PorB when embedded in OMV with different LOS phenotypes. In parallel, epidemiological evidence has shown that vaccination with OMV-based vaccines against N. meningitidis serogroup B (MenB) is associated with a reduced incidence of gonococcal infection, despite these vaccines being originally designed to target meningococci. This observation has triggered various studies aimed at identifying the molecular basis of this shared antigenicity between the two species. For the experiment presented in this manuscript, sera from MenB vaccinees were screened to isolate monoclonal antibodies (mAbs) capable of binding gonococcus. Most of these antibodies were directed either against LOS or PorB, which was identified as a shared and immunogenic target between N. meningitidis and N. gonorrhoeae. Among anti-PorB antibodies, mAb 01K12 showed the highest bactericidal activity against gonococcus. Thanks to HDX-MS, the epitope recognized by mAb 01K12 was mapped on both meningococcal and gonococcal PorB embedded in detergent extracted outer membrane vesicles (dOMVs). In summary, this work highlights the complex interplay between saccharide and protein antigens in OMV-based vaccines and reveals how LOS structural variation can influence protein antigens accessibility to the immune system and antibody generation. These findings contribute to the knowledge to support the rational design of next-generation vaccines and immunotherapeutics for multidrug-resistant N. gonorrhoeae.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/244317