Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG⁺ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines. Significance Our results suggest a rational way of designing and developing an improved typhoid conjugate vaccine and, by extension, to conjugate vaccines in general: first, modify a T-independent polysaccharide so that it no longer induces a T-independent response, then conjugate the polysaccharide to a suitable carrier protein restoring immunogenicity, thus creating a pure T-dependent antigen that induces a strongly boostable and long-lived response at an early age.

Shigellosis is a leading cause of diarrheal disease in low-middle-income countries (LMICs). Effective vaccines will help to reduce the disease burden, exacerbated by increasing antibiotic resistance, in the most susceptible population represented by young children. A challenge for a broadly protective vaccine against shigellosis is to cover the most epidemiologically relevant serotypes among >50 Shigella serotypes circulating worldwide. The GMMA platform has been proposed as an innovative delivery system for Shigella O-antigens, and we have developed a 4-component vaccine against S. sonnei, S. flexneri 1b, 2a and 3a identified among the most prevalent Shigella serotypes in LMICs. Driven by the immunogenicity results obtained in clinic with a first-generation mono-component vaccine, a new S. sonnei GMMA construct was generated and combined with three S. flexneri GMMA in a 4-component Alhydrogel formulation (altSonflex1-2-3). This formulation was highly immunogenic, with no evidence of negative antigenic interference in mice and rabbits. The vaccine induced bactericidal antibodies also against heterologous Shigella strains carrying O-antigens different from those included in the vaccine. The Monocyte Activation Test used to evaluate the potential reactogenicity of the vaccine formulation revealed no differences compared to the S. sonnei mono-component vaccine, shown to be safe in several clinical trials in adults. A GLP toxicology study in rabbits confirmed that the vaccine was well tolerated. The preclinical study results support the clinical evaluation of altSonflex1-2-3 in healthy populations, and a phase 1–2 clinical trial is currently ongoing.

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4-N-acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for Shigella sonnei O-antigen, Streptococcus pneumoniae serotype 12F, and Staphylococcus aureus types 5 and 8 capsular polysaccharides.

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.

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM197, Tetanus Toxoid and Diphtheria Toxoid.