Technology platforms are an important strategy to facilitate the design, development and implementation of vaccines to combat high-burden diseases that are still a threat for human populations, especially in low- and middle-income countries, and to address the increasing number and global distribution of pathogens resistant to antimicrobial drugs. Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles derived from engineered Gram-negative bacteria, represent an attractive technology to design affordable vaccines. Here, we show that GMMA, decorated with heterologous polysaccharide or protein antigens, leads to a strong and effective antigen-specific humoral immune response in mice. Importantly, GMMA promote enhanced immunogenicity compared to traditional formulations (e.g., recombinant proteins and glycoconjugate vaccines), without negative impact to the anti-GMMA immune response. Our findings support the use of GMMA as a “plug and play” technology for the development of effective combination vaccines targeting different bugs at the same time.

No commercial vaccine is yet available against Group A Streptococcus (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM₁₉₇ conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM₁₉₇ as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.

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.

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM₁₉₇; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM₁₉₇ in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM₁₉₇, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM₁₉₇. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.

Glycoconjugation is a well-established technology for vaccine development: linkage of the polysaccharide (PS) antigen to an appropriate carrier protein overcomes the limitations of PS T-independent antigens, making them effective in infants and providing immunological memory. Glycoconjugate vaccines have been successful in reducing the burden of different diseases globally. However, many pathogens still require a vaccine, and many of them display a variety of glycans on their surface that have been proposed as key antigens for the development of high-valency glycoconjugate vaccines. CDAP chemistry represents a generic conjugation strategy that is easily applied to PS with different structures. This chemistry utilizes common groups to a large range of PS and proteins, e.g., hydroxyl groups on the PS and amino groups on the protein. Here, new fast analytical tools to study CDAP reaction have been developed, and reaction conditions for PS activation and conjugation have been extensively investigated. Mathematical models have been built to identify reaction conditions to generate conjugates with wanted characteristics and successfully applied to a large number of bacterial PSs from different pathogens, e.g., Klebsiella pneumoniae, Salmonella Paratyphi A, Salmonella Enteritidis, Salmonella Typhimurium, Shighella sonnei and Shigella flexneri. Furthermore, using Salmonella Paratyphi A O-antigen and CRM₁₉₇ as models, a design of experiment approach has been used to study the impact of conjugation conditions and conjugate features on immunogenicity in rabbits. The approach used can be rapidly extended to other PSs and accelerate the development of high-valency glycoconjugate vaccines.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.