Toward a Shigella Vaccine: Opportunities and Challenges to Fight an Antimicrobial-Resistant Pathogen.

Shigellosis causes more than 200,000 deaths worldwide and most of this burden falls on Low- and Middle-Income Countries (LMICs), with a particular incidence in children under 5 years of age. In the last decades, Shigella has become even more worrisome because of the onset of antimicrobial-resistant strains (AMR). Indeed, the WHO has listed Shigella as one of the priority pathogens for the development of new interventions. To date, there are no broadly available vaccines against shigellosis, but several candidates are being evaluated in preclinical and clinical studies, bringing to light very important data and information. With the aim to facilitate the understanding of the state-of-the-art of Shigella vaccine development, here we report what is known about Shigella epidemiology and pathogenesis with a focus on virulence factors and potential antigens for vaccine development. We discuss immunity after natural infection and immunization. In addition, we highlight the main characteristics of the different technologies that have been applied for the development of a vaccine with broad protection against Shigella.

Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat.

Klebsiella pneumoniae (Kp) is an opportunistic pathogen and the leading cause of healthcare-associated infections, mostly affecting subjects with compromised immune systems or suffering from concurrent bacterial infections. However, the dramatic increase in hypervirulent strains and the emergence of new multidrug-resistant clones resulted in Kp occurrence among previously healthy people and in increased morbidity and mortality, including neonatal sepsis and death across low- and middle-income countries. As a consequence, carbapenem-resistant and extended spectrum ß-lactamase-producing Kp have been prioritized as a critical anti-microbial resistance threat by the World Health Organization and this has renewed the interest of the scientific community in developing a vaccine as well as treatments alternative to the now ineffective antibiotics. Capsule polysaccharide is the most important virulence factor of Kp and plays major roles in the pathogenesis but its high variability (more than 100 different types have been reported) makes the identification of a universal treatment or prevention strategy very challenging. However, less variable virulence factors such as the O-Antigen, outer membrane proteins as fimbriae and siderophores might also be key players in the fight against Kp infections. Here, we review elements of the current status of the epidemiology and the molecular pathogenesis of Kp and explore specific bacterial antigens as potential targets for both prophylactic and therapeutic solutions.

Effect of O-Antigen Chain Length Regulation on the Immunogenicity of Shigella and Salmonella Generalized Modules for Membrane Antigens (GMMA).

Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, Shigella and Salmonella GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.

Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Bordetella pertussis Adhesion to Epithelial Cells.

Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV)1 as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg- virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro Four out of the selected proteins conferred adhesive ability to E. coli Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance.

A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

From an in vivo to an in vitro relative potency (IVRP) assay to fully characterize a multicomponent O-antigen based vaccine against Shigella.

Outer membrane vesicles (OMV) represent an innovative platform for the design of polysaccharide based vaccines. Generalized Modules for Membrane Antigens (GMMA), OMV released from engineered Gram-negative bacteria, have been proposed for the delivery of the O-Antigen, key target for protective immunity against several pathogens including Shigella. altSonflex1-2-3 is a GMMA based vaccine, including S. sonnei and S. flexneri 1b, 2a and 3a O-Antigens, with the aim to elicit broad protection against the most prevalent Shigella serotypes, especially affecting children in low-middle income countries. Here we developed an In Vitro Relative Potency assay, based on recognition of O-Antigen by functional monoclonal antibodies selected to bind the key epitopes of the different O-Antigen active ingredients, directly applied to our Alhydrogel-formulated vaccine. Heat-stressed altSonflex1-2-3 formulations were generated and extensively characterized. The impact of detected biochemical changes in in vivo and in vitro potency assays was assessed. The overall results showed how the in vitro assay can replace the use of animals, overcoming the inherently high variability of in vivo potency studies. The entire panel of physico-chemical methods developed will contribute to detect suboptimal batches and will be valuable to perform stability studies. The work on Shigella vaccine candidate can be easily extended to other O-Antigen based vaccines.

Antigen presentation by Follicular Dendritic cells to cognate B cells is pivotal for Generalised Modules for Membrane Antigens (GMMA) immunogenicity.

GMMA has been proposed as a potent technology platform for the design of safe, effective and affordable vaccines. As GMMA are vesicles blebbing out of the outer membrane of Gram-negative bacteria, they contain lipopolysaccharides, lipoproteins and peptidoglycans that stimulate immune cells via Toll-like Receptors 4 (TLR4) or TLR2. Being basically nanoparticles, GMMA can be efficiently captured by Follicular Dendritic Cells (FDC) for antigen presentation to cognate B cells. GMMA have shown to be highly immunogenic in preclinical and clinical studies and the engagement of TLR4 and TLR2 or antigen presentation by FDC may have a prominent role in GMMA immunogenicity, which is well worth investigating. By using GMMA derived from Shigella sonnei and Salmonella Typhimurium, we show for the first time that the antigen presentation by FDC to cognate B cells plays a major role in the induction of an effective humoral immune response upon immunization with GMMA by using both models. The engagement of TLR4 is critical to elicit an optimal antibody production, but its effect on antibody functionality is dependent on GMMA type and is dispensable when immunizing with Alum adjuvant, whereas TLR2 does not have any role for GMMA immunogenicity. Our findings represent a substantial advancement of the knowledge on GMMA mode of action and shed a light on novel perspectives for the design of safer and more effective GMMA-based vaccines. ONE SENTENCE SUMMARY: The study demonstrated that the antigen presentation by FDC to cognate B cells plays a major role for GMMA immunogenicity.

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation.

The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen.

Impact of O-Acetylation on S. flexneri 1b and 2a O-Antigen Immunogenicity in Mice.

Shigellosis is a diarrheal disease caused prevalently by Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines.

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency.

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.

Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis.

Clostridium difficile is a Gram-positive, anaerobic bacterium and the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile modulates its transition from a motile to a sessile lifestyle through a mechanism of riboswitches regulated by cyclic diguanosine monophosphate (c-di-GMP). Previously described as a sortase substrate positively regulated by c-di-GMP, CD2831 was predicted to be a collagen-binding protein and thus potentially involved in sessility. By overexpressing CD2831 in C. difficile and heterologously expressing it on the surface of Lactococcus lactis, here we further demonstrated that CD2831 is a collagen-binding protein, able to bind to immobilized collagen types I, III and V as well as native collagen produced by human fibroblasts. We also observed that the overexpression of CD2831 raises the ability to form biofilm on abiotic surface in both C. difficile and L. lactis. Notably, we showed that CD2831 binds to the collagen-like domain of the human complement component C1q, suggesting a role in preventing complement cascade activation via the classical pathway. This functional characterization places CD2831 in the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMMs) family, a class of virulence factors with a dual role in adhesion to collagen-rich tissues and in host immune evasion by binding to human complement components.

Physiopathological roles of spontaneously released outer membrane vesicles of Bordetella pertussis.

AIM: Bordetella pertussis has been shown to release outer membrane vesicles (OMV) both in vitro and in vivo but little is known about their biological role during the initial phases of B. pertussis infection of the airways. RESULTS: We have demonstrated that OMV are released by B. pertussis in a human ciliated-airway cell model and purified vesicles can interact with host cells. Binding and uptake are strictly Bvg-regulated and OMV-associated pertussis toxin contributes to host-cell intoxication. Furthermore, we have shown that OMV act as iron-delivery systems complementing the B. pertussis growth defect in iron-limiting conditions. CONCLUSION: We have proved that OMV play different roles in B. pertussis physiopathology and we opened new perspectives to be further investigated.