Safety and Immunogenicity of a 4-Component Generalized Modules for Membrane Antigens Shigella Vaccine in Healthy European Adults: Randomized, Phase 1/2 Study.

BACKGROUND: We report data from stage 1 of an ongoing 2-staged, phase 1/2 randomized clinical trial with a 4-component generalized modules for membrane antigens-based vaccine against Shigella sonnei and Shigella flexneri 1b, 2a, and 3a (altSonflex1-2-3; GSK). METHODS: Europeans aged 18-50 years (N = 102) were randomized (2:1) to receive 2 injections of altSonflex1-2-3 or placebo at 3- or 6-month interval. Safety and immunogenicity were assessed at prespecified time points. RESULTS: The most common solicited administration-site event (until 7 days after each injection) and unsolicited adverse event (until 28 days after each injection) were pain (altSonflex1-2-3, 97.1%; placebo, 58.8%) and headache (32.4%; 23.5%), respectively. All serotype-specific functional IgG antibodies peaked 14-28 days after injection 1 and remained substantially higher than prevaccination at 3 or 6 months postvaccination; the second injection did not boost but restored the initial immune response. The highest seroresponse rates (≥4-fold increase in titers over baseline) were obtained against S. flexneri 2a (enzyme-linked immunosorbent assay [ELISA] after injection 1, 91.0%; after injection 2 [day 113; day 197], 100%; 97.0% and serum bactericidal activity [SBA] after injection 1, 94.4%; after injection 2, 85.7%; 88.9%) followed by S. sonnei (ELISA after injection 1, 77.6%; after injection 2, 84.6%; 78.8% and SBA after injection 1, 83.3%; after injection 2, 71.4%; 88.9%). Immune responses against S. flexneri 1b and S. flexneri 3a, as measured by both ELISA and SBA, were numerically lower compared to those against S. sonnei and S. flexneri 2a. CONCLUSIONS: No safety signals or concerns were identified. altSonflex1-2-3 induced functional serotype-specific immune responses, allowing further clinical development in the target population. Clinical Trials Registration . NCT05073003.

High-Throughput Luminescence-Based Serum Bactericidal Assay Optimization and Characterization to Assess Human Sera Functionality Against Multiple Shigella flexneri Serotypes.

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different Shigella serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread Shigella serotypes. The assay was previously developed and characterized for S. sonnei and S. flexneri 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (S. flexneri 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple Shigella serotypes, thus enabling analysis of the functional response induced by natural exposure to Shigella in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent Shigella serotypes in a complement-mediated fashion.

Streptococcus pyogenes Colonization in Children Aged 24-59 Months in the Gambia: Impact of Live Attenuated Influenza Vaccine and Associated Serological Responses.

BACKGROUND: Immunity to Streptococcus pyogenes in high burden settings is poorly understood. We explored S. pyogenes nasopharyngeal colonization after intranasal live attenuated influenza vaccine (LAIV) among Gambian children aged 24-59 months, and resulting serological response to 7 antigens. METHODS: A post hoc analysis was performed in 320 children randomized to receive LAIV at baseline (LAIV group) or not (control). S. pyogenes colonization was determined by quantitative polymerase chain reaction (qPCR) on nasopharyngeal swabs from baseline (day 0), day 7, and day 21. Anti-streptococcal IgG was quantified, including a subset with paired serum before/after S. pyogenes acquisition. RESULTS: The point prevalence of S. pyogenes colonization was 7%-13%. In children negative at day 0, S. pyogenes was detected at day 7 or 21 in 18% of LAIV group and 11% of control group participants (P = .12). The odds ratio (OR) for colonization over time was significantly increased in the LAIV group (day 21 vs day 0 OR, 3.18; P = .003) but not in the control group (OR, 0.86; P = .79). The highest IgG increases following asymptomatic colonization were seen for M1 and SpyCEP proteins. CONCLUSIONS: Asymptomatic S. pyogenes colonization appears modestly increased by LAIV, and may be immunologically significant. LAIV could be used to study influenza-S. pyogenes interactions. Clinical Trials Registration. NCT02972957.

Exploring the Role of GMMA Components in the Immunogenicity of a 4-Valent Vaccine against Shigella.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella.

Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197 Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with SalmonellaS Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.

Antibodies and Protection in Systemic Salmonella Infections: Do We Still Have More Questions than Answers?

Salmonella causes grave systemic infections in humans and other animals and provides a paradigm for other diseases in which the bacteria have both intracellular and extracellular lifestyles. New generations of vaccines rely on the essential contribution of the antibody responses for their protection. The quality, antigen specificity, and functions associated with antibody responses to this pathogen have been elusive for a long time. Recent approaches that combine studies in humans and genetically manipulated experimental models and that exploit awareness of the location and within-host life cycle of the pathogen are shedding light on how humoral immunity to Salmonella operates. However, this area of research remains full of controversy and discrepancies. The overall scenario indicates that antibodies are essential for resistance against systemic Salmonella infections and can express the highest protective function when operating in conjunction with cell-mediated immunity. Antigen specificity, isotype profile, Fc-gamma receptor usage, and complement activation are all intertwined factors that still arcanely influence antibody-mediated protection to Salmonella.

OMV Vaccines and the Role of TLR Agonists in Immune Response.

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.

The essential role of complement in antibody-mediated resistance to Salmonella.

Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long-lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc-gamma receptors (FcγRs) can potentially play a crucial role in IgG-mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene-targeted mice lacking either FcγRI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody-mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O-antigen in gene-targeted mice, we demonstrate that the complement system is essential for the IgG-mediated reduction of bacterial numbers in the tissues.

An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei.

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations.

Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

A flow cytometry-based assay to determine the ability of anti-Streptococcus pyogenes antibodies to mediate monocytic phagocytosis in human sera.

Streptococcus pyogenes, commonly referred to as Group A Streptococcus (Strep A), causes a spectrum of diseases, with the potential to progress into life-threatening illnesses and autoimmune complications. The escalating threat of antimicrobial resistance, stemming from the prevalent reliance on antibiotic therapies to manage Strep A infections, underscores the critical need for the development of disease control strategies centred around vaccination. Phagocytes play a critical role in controlling Strep A infections, and phagocytosis-replicating assays are essential for vaccine development. Traditionally, such assays have employed whole-blood killing or opsonophagocytic methods using HL-60 cells as neutrophil surrogates. However, assays mimicking Fcγ receptors- phagocytosis in clinical contexts are lacking. Therefore, here we introduce a flow cytometry-based method employing undifferentiated THP-1 cells as monocytic/macrophage model to swiftly evaluate the ability of human sera to induce phagocytosis of Strep A. We extensively characterize the assay's precision, linearity, and quantification limit, ensuring robustness. By testing human pooled serum, the assay proved to be suitable for the comparison of human sera's phagocytic capability against Strep A. This method offers a valuable complementary assay for clinical studies, addressing the gap in assessing FcγR-mediated phagocytosis. By facilitating efficient evaluation of Strep A -phagocyte interactions, it may contribute to elucidating the mechanisms required for the prevention of infections and inform the development of future vaccines and therapeutic advancements against Strep A infections.

Testing S. sonnei GMMA with and without Aluminium Salt-Based Adjuvants in Animal Models.

Shigellosis is one of the leading causes of diarrheal disease in low- and middle-income countries, particularly in young children, and is more often associated with antimicrobial resistance. Therefore, a preventive vaccine against shigellosis is an urgent medical need. We have proposed Generalised Modules for Membrane Antigens (GMMA) as an innovative delivery system for Shigella sonnei O-antigen, and an Alhydrogel formulation (1790GAHB) has been extensively tested in preclinical and clinical studies. Alhydrogel has been used as an adsorbent agent with the main purpose of reducing potential GMMA systemic reactogenicity. However, the immunogenicity and systemic reactogenicity of this GMMA-based vaccine formulated with or without Alhydrogel have never been compared. In this work, we investigated the potential adjuvant effect of aluminium salt-based adjuvants (Alhydrogel and AS37) on S. sonnei GMMA immunogenicity in mice and rabbits, and we found that S. sonnei GMMA alone resulted to be strongly immunogenic. The addition of neither Alhydrogel nor AS37 improved the magnitude or the functionality of vaccine-elicited antibodies. Interestingly, rabbits injected with either S. sonnei GMMA adsorbed on Alhydrogel or S. sonnei GMMA alone showed a limited and transient body temperature increase, returning to baseline values within 24 h after each vaccination. Overall, immunisation with unadsorbed GMMA did not raise any concern for animal health. We believe that these data support the clinical testing of GMMA formulated without Alhydrogel, which would allow for further simplification of GMMA-based vaccine manufacturing.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Development and characterization of a hemolysis inhibition assay to determine functionality of anti-Streptolysin O antibodies in human sera.

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies.

Development of a visual Adhesion/Invasion Inhibition Assay to assess the functionality of Shigella-specific antibodies.

Introduction: Shigella is the etiologic agent of a bacillary dysentery known as shigellosis, which causes millions of infections and thousands of deaths worldwide each year due to Shigella's unique lifestyle within intestinal epithelial cells. Cell adhesion/invasion assays have been extensively used not only to identify targets mediating host-pathogen interaction, but also to evaluate the ability of Shigella-specific antibodies to reduce virulence. However, these assays are time-consuming and labor-intensive and fail to assess differences at the single-cell level. Objectives and methods: Here, we developed a simple, fast and high-content method named visual Adhesion/Invasion Inhibition Assay (vAIA) to measure the ability of anti-Shigellaantibodies to inhibit bacterial adhesion to and invasion of epithelial cells by using the confocal microscope Opera Phenix. Results: We showed that vAIA performed well with a pooled human serum from subjects challenged with S. sonnei and that a specific anti-IpaD monoclonal antibody effectively reduced bacterial virulence in a dose-dependent manner. Discussion: vAIA can therefore inform on the functionality of polyclonal and monoclonal responses thereby supporting the discovery of pathogenicity mechanisms and the development of candidate vaccines and immunotherapies. Lastly, this assay is very versatile and may be easily applied to other Shigella species or serotypes and to different pathogens.

Qualification of an enzyme-linked immunosorbent assay for quantification of anti-Vi IgG in human sera.

Effective vaccines against Salmonella Typhi, targeting the Vi capsular polysaccharide, have been developed and are being introduced into routine immunization in endemic countries. Vi conjugated vaccines are also being tested in new multi-component vaccine formulations. Simple, high-throughput and cost-effective assays to quantify Vi-specific IgG in clinical sera are needed. In this study we present the development and qualification of a new anti-Vi ELISA with continuous readout, which expresses results as ELISA Unit/mL (EU/mL). We have qualified the assay in terms of precision, linearity and specificity, demonstrating performance in line with a commercially available anti-Vi ELISA. We have also calibrated the assay against the 16/138 anti-Vi international standard and established conversion factor between EU/mL and international units/mL, to allow comparability of results across studies. In summary, this new assay met all the suitability criteria and is being used to evaluate anti-Vi responses in clinical studies.

Modeling 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) Chemistry to Design Glycoconjugate Vaccines with Desired Structural and Immunological Characteristics.

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.

Putative correlates of protection against shigellosis assessing immunomarkers across responses to S. sonnei investigational vaccine.

Shigella spp. are a leading bacterial cause of diarrhea. No widely licensed vaccines are available and there is no generally accepted correlate of protection. We tested a S. sonnei Generalized Modules for Membrane Antigen (GMMA)-based vaccine (1790GAHB) in a phase 2b, placebo-controlled, randomized, controlled human infection model study (NCT03527173) enrolling healthy United States adults aged 18-50 years. We report analyses evaluating immune responses to vaccination, with the aim to identify correlates of risk for shigellosis among assessed immunomarkers. We found that 1790GAHB elicited S. sonnei lipopolysaccharide specific α4ß7+ immunoglobulin (Ig) G and IgA secreting B cells which are likely homing to the gut, indicating the ability to induce a mucosal in addition to a systemic response, despite parenteral delivery. We were unable to establish or confirm threshold levels that predict vaccine efficacy facilitating the evaluation of vaccine candidates. However, serum anti-lipopolysaccharide IgG and bactericidal activity were identified as potential correlates of risk for shigellosis.

Age-dependent acquisition of IgG antibodies to Shigella serotypes-a retrospective analysis of seroprevalence in Kenyan children with implications for infant vaccination.

Background: Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. Methods: We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. Results: A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Conclusion: Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.