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.

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.

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.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM₁₉₇), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM₁₉₇ glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

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.

Klebsiella pneumoniae is a leading cause of nosocomial infections, neonatal sepsis, and childhood mortality worldwide. A drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity, and the World Health Organization (WHO) has classified this as a critical-priority antimicrobial-resistant (AMR) pathogen. Recent advancements in developing vaccines against Klebsiella pneumoniae have highlighted the lack of standardized assays to evaluate immunogenicity, complicating comparison among different vaccines under development and the establishment of a serological threshold of risk reduction (SToRR). Here, we describe the development of a ten-plex multiplex assay to measure IgG against capsular polysaccharides (K2, K25, K102, K149), O antigens (O1v1, O1v2, O2v1, O2v2 and O5), and a conserved protein (MrkA). A standard curve was established by pooling human sera from naturally exposed subjects and then calibrated in terms of Relative Luminex Units/mL. The assay was fully characterized in terms of specificity, precision, linearity, and repeatability. This immunoassay demonstrates performance suitable for future clinical trials, as well as to perform sero-epidemiological studies to gain insights into naturally occurring immunity, potentially contributing to the establishment of a serological threshold of risk reduction against Klebsiella pneumoniae.