The 2022 Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference: Summary of breakout workshops.

The global public health nonprofit organization PATH hosted the third Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC, from November 29 to December 1, 2022. This international gathering focused on cutting-edge research related to the development of vaccines against neglected diarrheal pathogens including Shigella, enterotoxigenic Escherichia coli (ETEC), Campylobacter, and non-typhoidal Salmonella. In addition to the conference's plenary content, the agenda featured ten breakout workshops on topics of importance to the enteric vaccine field. This unique aspect of VASE Conferences allows focused groups of attendees to engage in in-depth discussions on subjects of interest to the enteric vaccine development community. In 2022, the workshops covered a range of topics. Two focused on the public health value of enteric vaccines, with one examining how to translate evidence into policy and the other on the value proposition of potential combination vaccines against bacterial enteric pathogens. Two more workshops explored new tools for the development and evaluation of vaccines, with the first on integrating antigen/antibody technologies for mucosal vaccine and immunoprophylactic development, and the second on adjuvants specifically for Shigella vaccines for children in low- and middle-income countries. Another pair of workshops covered the status of vaccines against two emerging enteric pathogens, Campylobacter and invasive non-typhoidal Salmonella. The remaining four workshops examined the assessment of vaccine impact on acute and long-term morbidity. These included discussions on the nature and severity of intestinal inflammation; cellular immunity and immunological memory in ETEC and Shigella infections; clinical and microbiologic endpoints for Shigella vaccine efficacy studies in children; and intricacies of protective immunity to enteric pathogens. This article provides a brief summary of the presentations and discussions at each workshop in order to share these sessions with the broader enteric vaccine field.

Controlled human infection trials: Legitimacy and conditions of implementation in France.

This round table is the result of an observation. The observation being that controlled human infection clinical trials (also called "infectious challenge" trials or "Controlled Human Infection Models", "CHIM") recommended or even encouraged in the context of vaccine developments in particular, are not carried out in France. However, there are no formal prohibitions within regulations or ethical principles, which point to the prior assessment of risks and benefits for individuals and for society. The participants in this Round Table thus wished to examine, through the prism of their respective disciplines, the scientific and medical relevance of conducting such trials in France and, if possible, to imagine the conditions under which they would be carried out, thus resulting in recommendations on (1) the advisability of their conduct in France (2), the conditions under which they would be implemented in terms of logistics and regulations, and (3) their social acceptability. The recommendations on which the participants of the Round Table came to an agreement are presented as the analysis progresses.

Further characterization of Shigella-specific (memory) B cells induced in healthy volunteer recipients of SF2a-TT15, a Shigella flexneri 2a synthetic glycan-based vaccine candidate.

Shigellosis is common worldwide, and it causes significant morbidity and mortality mainly in young children in low- and middle- income countries. To date, there are not broadly available licensed Shigella vaccines. A novel type of conjugate vaccine candidate, SF2a-TT15, was developed against S. flexneri serotype 2a (SF2a). SF2a-TT15 is composed of a synthetic 15mer oligosaccharide, designed to act as a functional mimic of the SF2a O-antigen and covalently linked to tetanus toxoid (TT). SF2a-TT15 was recently shown to be safe and immunogenic in a Phase 1 clinical trial, inducing specific memory B cells and sustained antibody response up to three years after the last injection. In this manuscript, we advance the study of B cell responses to parenteral administration of SF2a-TT15 to identify SF2a LPS-specific B cells (SF2a+ B cells) using fluorescently labeled bacteria. SF2a+ B cells were identified mainly within class-switched B cells (SwB cells) in volunteers vaccinated with SF2a-TT15 adjuvanted or not with aluminium hydroxide (alum), but not in placebo recipients. These cells expressed high levels of CXCR3 and low levels of CD21 suggesting an activated phenotype likely to represent the recently described effector memory B cells. IgG SF2a+ SwB cells were more abundant than IgA SF2a + SwB cells. SF2a+ B cells were also identified in polyclonally stimulated B cells (antibody secreting cells (ASC)-transformed). SF2a+ ASC-SwB cells largely maintained the activated phenotype (CXCR3 high, CD21 low). They expressed high levels of CD71 and integrin α4ß7, suggesting a high proliferation rate and ability to migrate to gut associated lymphoid tissues. Finally, ELISpot analysis showed that ASC produced anti-SF2a LPS IgG and IgA antibodies. In summary, this methodology confirms the ability of SF2a-TT15 to induce long-lived memory B cells, initially identified by ELISpots, which remain identifiable in blood up to 140 days following vaccination. Our findings expand and complement the memory B cell data previously reported in the Phase 1 trial and provide detailed information on the immunophenotypic characteristics of these cells. Moreover, this methodology opens the door to future studies at the single-cell level to better characterize the development of B cell immunity to Shigella.

A Shigella flexneri 2a synthetic glycan-based vaccine induces a long-lasting immune response in adults.

Shigella is a leading cause of moderate to severe diarrhea worldwide and of diarrhea-associated deaths in children under 5 years of age in low-and middle-income countries. A vaccine against shigellosis is in high demand. SF2a-TT15, a synthetic carbohydrate-based conjugate vaccine candidate against Shigella flexneri 2a (SF2a) was found safe and strongly immunogenic in adult volunteers. Here, SF2a-TT15 at 10 µg oligosaccharide (OS) vaccine dose is shown to induce a sustained immune response in magnitude and functionality in the majority of volunteers followed up 2 and 3 years post-vaccination. High levels of either one of the humoral parameters as well as the number of specific-IgG memory B-cells determined 3 months after vaccination were good predictors of the durability of the immune response. This study is the first to examine the long-term durability of antibody functionality and memory B-cell response induced by a Shigella vaccine candidate.

The First-in-Human Synthetic Glycan-Based Conjugate Vaccine Candidate against Shigella.

Shigella, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting Shigella flexneri 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.

Toward a Multivalent Synthetic Oligosaccharide-Based Conjugate Vaccine against Shigella: State-of-the-Art for a Monovalent Prototype and Challenges.

This review focuses on the molecular glycovaccine concept, a promising option to develop a Shigella glycoconjugate vaccine. Subsequent to original developments involving, as main vaccine component, the detoxified Shigella lipopolysaccharide randomly conjugated at multiple sites to a carrier protein, novelty stems from the use of rationally designed, well-defined chemically synthesized oligosaccharide haptens conceived as functional surrogates of the main surface antigen, linked via single-point attachment onto a carrier. The concept and design of such a fine-tuned Shigella glycovaccine are presented by way of SF2a-TT15, a neoglycoprotein featuring a synthetic 15-mer oligosaccharide, which constitutes an original vaccine prototype targeting Shigella flexneri 2a, one of the predominant circulating strains in endemic settings. The clinical testing of SF2a-TT15 is summarized with the first-in-human phase I trial in young healthy adults showing a good safety profile and tolerability, while inducing bactericidal antibodies towards S. flexneri 2a bacteria. The proof-of-concept of this novel approach being established, an ongoing phase IIa clinical study in the nine-month-old infant target population in endemic area was launched, which is also outlined. Lastly, some challenges to move forward this original approach toward a multivalent cost-effective Shigella synthetic glycan conjugate vaccine are introduced.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study.

BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.

Shigella impairs human T lymphocyte responsiveness by hijacking actin cytoskeleton dynamics and T cell receptor vesicular trafficking.

Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.

Role of antigen specific T and B cells in systemic and mucosal immune responses in ETEC and Shigella infections, and their potential to serve as correlates of protection in vaccine development.

The generation of robust systemic and mucosal antibody and cell-mediated immune (CMI) responses that are protective, long-lasting, and can quickly be recalled upon subsequent re-exposure to the cognate antigen is the key to the development of effective vaccine candidates. These responses, whether they represent mechanistic or non-mechanistic immunological correlates of protection, usually entail the activation of T cell memory and effector subsets (T-CMI) and induction of long-lasting memory B cells. However, for ETEC and Shigella, the precise role of these key immune cells in primary and secondary (anamnestic) immune responses remains ill-defined. A workshop to address immune correlates for ETEC and Shigella, in general, and to elucidate the mechanistic role of T-cell subsets and B-cells, both systemically and in the mucosal microenvironment, in the development of durable protective immunity against ETEC and Shigella was held at the recent 2nd Vaccines against Shigella and ETEC (VASE) conference in June 2018. This report is a summary of the presentations and the discussion that ensued at the workshop.

Shigella-mediated immunosuppression in the human gut: subversion extends from innate to adaptive immune responses.

The enteropathogen, Shigella, is highly virulent and remarkably adjusted to the intestinal environment of its almost exclusive human host. Key for Shigella pathogenicity is the injection of virulence effectors into the host cell via its type three secretion system (T3SS), initiating disease onset and progression by the vast diversity of the secreted T3SS effectors and their respective cellular targets. The multifaceted modulation of host signaling pathways exerted by Shigella T3SS effectors, which include the subversion of host innate immune defenses and the promotion of intracellular bacterial survival and dissemination, have been extensively reviewed in the recent past. This review focuses on the human species specificity of Shigella by discussing some possible evasion mechanisms towards the human, but not non-human or rodent gut innate defense barrier, leading to the lack of a relevant animal infection model. In addition, subversion mechanisms of the adaptive immune response are highlighted summarizing research advances of the recent years. In particular, the new paradigm of Shigella pathogenicity constituted of invasion-independent T3SS effector-mediated targeting of activated, human lymphocytes is discussed. Along with consequences on vaccine development, these findings offer new directions for future research endeavors towards a better understanding of immunity to Shigella infection.

Glycan-Glycan Interaction Determines Shigella Tropism toward Human T Lymphocytes.

Direct interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis.IMPORTANCE Glycosylation of host cell surface varies with species and location in the body, thus contributing to species specificity and tropism of microorganisms. Cross talk by Shigella, the Gram-negative enteroinvasive bacterium responsible for bacillary dysentery, with its exclusively human host has been extensively studied. However, the molecular determinants of the step of binding to host cells are poorly defined. Taking advantage of the observation that human-activated CD4+ T lymphocytes, but not nonactivated cells, are targets of Shigella, we succeeded in rendering the refractory cells susceptible to targeting upon loading of their plasma membrane with sialylated glycosphingolipids (gangliosides) that are abundantly present on activated cells. We show that interactions between the sugar polar part of gangliosides and the polysaccharide moiety of Shigella lipopolysaccharide (LPS) promote bacterial binding, which results in the injection of effectors via the type III secretion system. Whereas LPS interaction with gangliosides was proposed long ago and recently extended to a large variety of glycans, our findings reveal that such glycan-glycan interactions are critical for Shigella pathogenesis by driving selective interactions with host cells, including immune cells.

Host Cell Targeting by Enteropathogenic Bacteria T3SS Effectors.

Microbial pathogens possess a diversity of weapons that disrupt host homeostasis and immune defenses, thus resulting in the establishment of infection. The best-characterized system mediating bacterial protein delivery into target eukaryotic cells is the type III secretion system (T3SS) expressed by Gram-negative bacteria, including the human enteric pathogens Shigella, Salmonella, Yersinia, and enteropathogenic/enterohemorragic Escherichia coli (EPEC/EHEC). The emerging global view is that these T3SS-bearing pathogens share similarities in their ability to target key cellular pathways such as the cell cytoskeleton, trafficking, cell death/survival, and the NF-κB and MAPK signaling pathways. In particular, multiple host proteins are targeted in a given pathway, and different T3SS effectors from various pathogens share functional similarities.

Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using ß-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes.

The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.

Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri†3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -ß-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

A Synthetic Carbohydrate Conjugate Vaccine Candidate against Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity.

Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.

Draft genomes of Shigella strains used by the STOPENTERICS consortium.

BACKGROUND: Despite a significant global burden of disease, there is still no vaccine against shigellosis widely available. One aim of the European Union funded STOPENTERICS consortium is to develop vaccine candidates against Shigella. Given the importance of translational vaccine coverage, here we aimed to characterise the Shigella strains being used by the consortium by whole genome sequencing, and report on the stability of strains cultured in different laboratories or through serial passage. METHODS: We sequenced, de novo assembled and annotated 20 Shigella strains being used by the consortium. These comprised 16 different isolates belonging to 7 serotypes, and 4 derivative strains. Derivative strains from common isolates were manipulated in different laboratories or had undergone multiple passages in the same laboratory. Strains were mapped against reference genomes to detect SNP variation and phylogenetic analysis was performed. RESULTS: The genomes assembled into similar total lengths (range 4.14-4.83 Mbp) and had similar numbers of predicted coding sequences (average of 4,400). Mapping analysis showed the genetic stability of strains through serial passages and culturing in different laboratories, as well as varying levels of similarity to published reference genomes. Phylogenetic analysis revealed the presence of three main clades among the strains and published references, one containing the Shigella flexneri serotype 6 strains, a second containing the remaining S. flexneri serotypes and a third comprised of Shigella sonnei strains. CONCLUSIONS: This work increases the number of the publically available Shigella genomes available and specifically provides information on strains being used for vaccine development by STOPENTERICS. It also provides information on the variability among strains maintained in different laboratories and through serial passage. This work will guide the selection of strains for further vaccine development.