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Klebsiella pneumoniae causes community- and healthcare-associated infections in children and adults. Globally in 2019, an estimated 1.27 million (95% Uncertainty Interval [UI]: 0.91-1.71) and 4.95 million (95% UI: 3.62-6.57) deaths were attributed to and associated with bacterial antimicrobial resistance (AMR), respectively. K. pneumoniae was the second leading pathogen in deaths attributed to AMR resistant bacteria. Furthermore, the rise of antimicrobial resistance in both community- and hospital-acquired infections is a concern for neonates and infants who are at high risk for invasive bacterial disease. There is a limited antibiotic pipeline for new antibiotics to treat multidrug resistant infections, and vaccines targeted against K. pneumoniae are considered to be of priority by the World Health Organization. Vaccination of pregnant women against K. pneumoniae could reduce the risk of invasive K.pneumoniae disease in their young offspring. In addition, vulnerable children, adolescents and adult populations at risk of K. pneumoniae disease with underlying diseases such as immunosuppression from underlying hematologic malignancy, chemotherapy, patients undergoing abdominal and/or urinary surgical procedures, or prolonged intensive care management are also potential target groups for a K. pneumoniae vaccine. A 'Vaccine Value Profile' (VVP) for K.pneumoniae, which contemplates vaccination of pregnant women to protect their babies from birth through to at least three months of age and other high-risk populations, provides a high-level, holistic assessment of the available information to inform the potential public health, economic and societal value of a pipeline of K. pneumoniae vaccines and other preventatives and therapeutics. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public-private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the WHO. All contributors have extensive expertise on various elements of the K.pneumoniae VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ziyaad Dangor reports financial support was provided by Bill & Melinda Gates Foundation. Ziyaad Dangor reports financial support was provided by World Health Organization. If there are other authors, they 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 © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria engineered to provide an over-vesiculating phenotype, which represent an attractive platform for the design of affordable vaccines. GMMA can be further genetically manipulated to modulate the risk of systemic reactogenicity and to act as delivery system for heterologous polysaccharide or protein antigens. GMMA are able to induce strong immunogenicity and protection in animal challenge models, and to be well-tolerated and immunogenic in clinical studies. The high immunogenicity could be ascribed to their particulate size, to their ability to present to the immune system multiple antigens in a natural conformation which mimics the bacterial environment, as well as to their intrinsic self-adjuvanticity. However, GMMA mechanism of action and the role in adjuvanticity are still unclear and need further investigation. In this review, we discuss progresses in the development of the GMMA vaccine platform, highlighting successful applications and identifying knowledge gaps and potential challenges., Competing Interests: This work was undertaken at the request of and sponsored by GlaxoSmithKline Biologicals SA. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. All authors are employed by the GSK group of companies., (Copyright © 2021 Mancini, Micoli, Necchi, Pizza, Berlanda Scorza and Rossi.)

Shigella is the second most deadly diarrheal disease among children under five years of age, after rotavirus, with high morbidity and mortality in developing countries. Currently, no vaccine is widely available, and the increasing levels of multidrug resistance make Shigella a high priority for vaccine development. The single-component candidate vaccine against Shigella sonnei (1790GAHB), developed using the GMMA technology, contains the O antigen (OAg) portion of lipopolysaccharide (LPS) as active moiety. The vaccine was well tolerated and immunogenic in early-phase clinical trials. In a phase 1 placebo-controlled dose escalation trial in France (NCT02017899), three doses of five different vaccine formulations (0.06/1, 0.3/5, 1.5/25, 3/50, 6/100 µg of OAg/protein) were administered to healthy adults. In the phase 1 extension trial (NCT03089879), conducted 2-3 years following the parent study, primed individuals who had undetectable antibody levels before the primary series received a 1790GAHB booster dose (1.5/25 µg OAg/protein). Controls were unprimed participants immunized with one 1790GAHB dose. The current analysis assessed the functionality of sera collected from both studies using a high-throughput luminescence-based serum bactericidal activity (SBA) assay optimized for testing human sera. Antibodies with complement-mediated bactericidal activity were detected in vaccinees but not in placebo recipients. SBA titers increased with OAg dose, with a persistent response up to six months after the primary vaccination with at least 1.5/25 µg of OAg/protein. The booster dose induced a strong increase of SBA titers in most primed participants. Correlation between SBA titers and anti- S. sonnei LPS serum immunoglobulin G levels was observed. Results suggest that GMMA is a promising OAg delivery system for the generation of functional antibody responses and persistent immunological memory., Competing Interests: FM, OR, VC, ASS, MA, UN, EM, RR, LM, FN, and AP are employees of the GSK group of companies and RR, LM, and AP hold shares in the GSK group of companies. AS was an employee of the GSK group of companies at the time of the study and holds shares in the GSK group of companies. FM and AS report grant from the Bill and Melinda Gates Foundation during the conduct of the study. LM reports grant from EU FP7 STOPENTERICS during the conduct of the study and from the Bill and Melinda Gates Foundation and Wellcome Trust outside the submitted work. LM and AS are inventors of patents owned by the GSK group of companies and relevant to Shigella vaccine. OL’s institution received grant from the GSK group of companies for conducting the study. All authors have no non-financial relationships and activities., (Copyright © 2021 Micoli, Rossi, Conti, Launay, Sciré, Aruta, Nakakana, Marchetti, Rappuoli, Saul, Martin, Necchi and Podda.)

The use of antibiotics has enabled the successful treatment of bacterial infections, saving the lives and improving the health of many patients worldwide. However, the emergence and spread of antimicrobial resistance (AMR) has been highlighted as a global threat by different health organizations, and pathogens resistant to antimicrobials cause substantial morbidity and death. As resistance to multiple drugs increases, novel and effective therapies as well as prevention strategies are needed. In this Review, we discuss evidence that vaccines can have a major role in fighting AMR. Vaccines are used prophylactically, decreasing the number of infectious disease cases, and thus antibiotic use and the emergence and spread of AMR. We also describe the current state of development of vaccines against resistant bacterial pathogens that cause a substantial disease burden both in high-income countries and in low- and medium-income countries, discuss possible obstacles that hinder progress in vaccine development and speculate on the impact of next-generation vaccines against bacterial infectious diseases on AMR.

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) have received increased attention in recent years as a vaccine platform against bacterial pathogens. OMV from Neisseria meningitidis serogroup B have been extensively explored. Following the success of the MeNZB OMV vaccine in controlling an outbreak of N. meningitidis B in New Zealand, additional research and development resulted in the licensure of the OMV-containing four-component 4CMenB vaccine, Bexsero. This provided broader protection against multiple meningococcal B strains. Advances in the field of genetic engineering have permitted further improvements in the platform resulting in increased yields, reduced endotoxicity and decoration with homologous and heterologous antigens to enhance immuno genicity and provide broader protection. The OMV vaccine platform has been extended to many other pathogens. In this review, we discuss progress in the development of the OMV vaccine delivery platform, highlighting successful applications, together with potential challenges and gaps., (Copyright © 2020. Published by Elsevier Ltd.)

Glycoconjugate vaccines are among the safest and most successful vaccines developed during the past 30 years. Since the first semisynthetic chemical conjugate vaccine licensed in the 1980's to protect human against Haemophilus influenzae type b infection, conjugate vaccines against Neisseria meningitidis and Streptococcus pneumoniae have been developed and registered using the same approach (i.e. bacterial growth to produce capsular polysaccharide antigen and chemical coupling to carrier protein). Other types of conjugate vaccines have been recently developed and tested in clinical trials, prepared by coupling chemically synthesized oligosaccharides to proteins, by engineering Escherichia coli to directly produce bioconjugates or by delivering the native carbohydrate antigen in engineered membrane vesicles (i.e. Generalized Modules for Membrane Antigens, GMMA). Through this review, the reader will have an insight regarding the history and the characteristics of different types of conjugate vaccines, and the attributes that might affect their immunogenicity and their potential for future applications., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Outer Membrane Vesicles (OMVs) are bacterial nanoparticles that are spontaneously released during growth both in vitro and in vivo by Gram-negative bacteria. They are spherical, bilayered membrane nanostructures that contain many components found within the external surface of the parent bacterium. Naturally, OMVs serve the bacteria as a mechanism to deliver DNA, RNA, proteins, and toxins, as well as to promote biofilm formation and remodel the outer membrane during growth. On the other hand, as OMVs possess the optimal size to be uptaken by immune cells, and present a range of surface-exposed antigens in native conformation and Toll-like receptor (TLR) activating components, they represent an attractive and powerful vaccine platform able to induce both humoral and cell-mediated immune responses. This work reviews the TLR-agonists expressed on OMVs and their capability to trigger individual TLRs expressed on different cell types of the immune system, and then focuses on their impact on the immune responses elicited by OMVs compared to traditional vaccines.

Introduction : Over the last decades, glycoconjugate vaccines have been proven to be a successful strategy to prevent infectious diseases. Many diseases remain to be controlled, especially in developing countries, and emerging antibiotic-resistant bacteria present an alarming public-health threat. The increasing complexity of future vaccines, and the need to accelerate development processes have triggered the development of faster approaches to glycoconjugate vaccines design. Areas covered : This review provides an overview of recent progress in glycoconjugation technologies toward faster vaccine design. Expert opinion : Among the different emerging approaches, glycoengineering has the potential to combine glycan assembly and conjugation to carrier systems (such as proteins or outer membrane vesicles) in one step, resulting in a simplified manufacturing process and fewer analytical controls. Chemical and enzymatic strategies, and their automation can facilitate glycoepitope identification for vaccine design. Other approaches, such as the liposomal encapsulation of polysaccharides, potentially enable fast and easy combination of numerous antigens in the same formulation. Additional progress is envisaged in the near future, and some of these systems still need to be further validated in humans. In parallel, new strategies are needed to accelerate the vaccine development process, including the associated clinical trials, up to vaccine release onto the market.

Cell surface carbohydrates have been proven optimal targets for vaccine development. Conjugation of polysaccharides to a carrier protein triggers a T-cell-dependent immune response to the glycan moiety. Licensed glycoconjugate vaccines are produced by chemical conjugation of capsular polysaccharides to prevent meningitis caused by meningococcus, pneumococcus and Haemophilus influenzae type b. However, other classes of carbohydrates (O-antigens, exopolysaccharides, wall/teichoic acids) represent attractive targets for developing vaccines. Recent analysis from WHO/CHO underpins alarming concern toward antibiotic-resistant bacteria, such as the so called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) and additional pathogens such as Clostridium difficile and Group A Streptococcus. Fungal infections are also becoming increasingly invasive for immunocompromised patients or hospitalized individuals. Other emergencies could derive from bacteria which spread during environmental calamities (Vibrio cholerae) or with potential as bioterrorism weapons (Burkholderia pseudomallei and mallei, Francisella tularensis). Vaccination could aid reducing the use of broad-spectrum antibiotics and provide protection by herd immunity also to individuals who are not vaccinated.This review analyzes structural and functional differences of the polysaccharides exposed on the surface of emerging pathogenic bacteria, combined with medical need and technological feasibility of corresponding glycoconjugate vaccines.

Abstract: An efficacious, low cost vaccine against typhoid fever, especially for young children, would make a major impact on disease burden in developing countries. The virulence capsular polysaccharide of Salmonella Typhi (Vi) coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) has been shown to be highly efficacious. We investigated the use of carrier proteins included in infant vaccines, standardized the conjugation process and developed key assays required for routine lot release at production scale. Vi from a BSL1 organism, Citrobacter freundii, strain WR7011, was used as an alternative to Vi from S. Typhi. We showed that Vi conjugated to CRM197, a non-toxic mutant of diphtheria toxin, widely used in commercial vaccines, was produced at high yield. Vi-CRM197 proved immunogenic in animal studies, even without adjuvant. Thus, Vi-CRM197 appears to be a suitable candidate for the development of a commercially viable, effective typhoid vaccine for developing countries. [ABSTRACT FROM AUTHOR]

Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S . Typhimurium and S . Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium Δ tolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications. In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS. [ABSTRACT FROM AUTHOR]

Glycoconjugate vaccines are made of carbohydrate antigens covalently bound to a carrier protein to enhance their immunogenicity. Among the different carrier proteins tested in preclinical and clinical studies, five have been used so far for licensed vaccines: Diphtheria and Tetanus toxoids, the non-toxic mutant of diphtheria toxin CRM 197 , the outer membrane protein complex of Neisseria meningitidis serogroup B and the Protein D derived from non-typeable Haemophilus influenzae . Availability of novel carriers might help to overcome immune interference in multi-valent vaccines containing several polysaccharide-conjugate antigens, and also to develop vaccines which target both protein as well saccharide epitopes of the same pathogen. Accordingly we have conducted a study to identify new potential carrier proteins. Twenty-eight proteins, derived from different bacteria, were conjugated to the model polysaccharide Laminarin and tested in mice for their ability in inducing antibodies against the carbohydrate antigen and eight of them were subsequently tested as carrier for serogroup meningococcal C oligosaccharides. Four out of these eight were able to elicit in mice satisfactory anti meningococcal serogroup C titers. Based on immunological evaluation, the Streptococcus pneumoniae protein spr96/2021 was successfully evaluated as carrier for serogroups A, C, W, Y and X meningococcal capsular saccharides. [ABSTRACT FROM AUTHOR]

Salmonella paratyphi A is increasingly recognized as a common cause of enteric fever cases and there are no licensed vaccines against this infection. Antibodies directed against the O-polysaccharide of the lipopolysaccharide of Salmonella are protective and conjugation of the O-polysaccharide to a carrier protein represents a promising strategy for vaccine development. O-Acetylation of S. paratyphi A O-polysaccharide is considered important for the immunogenicity of S. paratyphi A conjugate vaccines. Here, as part of a programme to produce a bivalent conjugate vaccine against both S. typhi and S. paratyphi A diseases, we have fully elucidated the O-polysaccharide structure of S . paratyphi A by use of HPLC–SEC, HPAEC–PAD/CD, GLC, GLC–MS, 1D and 2D-NMR spectroscopy. In particular, chemical and NMR studies identified the presence of O -acetyl groups on C-2 and C-3 of rhamnose in the lipopolysaccharide repeating unit, at variance with previous reports of O-acetylation at a single position. Moreover HR-MAS NMR analysis performed directly on bacterial pellets from several strains of S. paratyphi A also showed O-acetylation on C-2 and C-3 of rhamnose, thus this pattern is common and not an artefact from O-polysaccharide purification. Conjugation of the O-polysaccharide to the carrier protein had little impact on O-acetylation and therefore should not adversely affect the immunogenicity of the vaccine. [ABSTRACT FROM AUTHOR]