Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge.

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCV-Sf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

Limited O-specific polysaccharide (OSP)-specific functional antibody responses in young children with Shigella infection in Bangladesh.

Shigellosis is the second leading cause of diarrheal death in children younger than five years of age globally. At present, there is no broadly licensed vaccine against shigella infection. Previous vaccine candidates have failed at providing protection for young children in endemic settings. Improved understanding of correlates of protection against Shigella infection and severe shigellosis in young children living in endemic settings is needed. Here, we applied a functional antibody profiling approach to define Shigella-specific antibody responses in young children versus older individuals with culture-confirmed shigellosis in Bangladesh, a Shigella endemic area. We analyzed Shigella-specific antibody isotypes, FcR binding and antibody-mediated innate immune cell activation in longitudinal serum samples collected at clinical presentation and up to 1 year later. We found that higher initial Shigella O-specific polysaccharide (OSP)-specific and protein-specific IgG and FcγR binding levels correlated with less severe disease regardless of patient age, but that individuals under 5 years of age developed a less prominent class switched, FcR-binding, functional and durable antibody response against both OSP and protein Shigella antigens than older individuals. Focusing on the largest cohort, we found that functional S. flexneri 2a OSP-specific responses were significantly induced only in individuals over age 5 years, and that these responses promoted monocyte phagocytosis and activation. Our findings suggest that in a Shigella endemic region, young children with shigellosis harbor a functional antibody response that fails to maximally activate monocytes; such a response may be important in facilitating subsequent innate cell clearance of Shigella, especially via recruitment and activation of polymorphonuclear cells capable of directly killing Shigella.

Determinants of immune responses predictive of protection against shigellosis in an endemic zone: a systems analysis of antibody profiles and function.

BACKGROUND: Shigella is the third leading global cause of moderate or severe diarrhoea among children younger than 5 years globally, and is the leading cause in children aged 24-59 months. The mechanism of protection against Shigella infection and disease in endemic areas is uncertain. We aimed to compare the Shigella-specific antibody responses in individuals living in Shigella-endemic and non-endemic areas, and to identify correlates of protection in a Shigella-endemic location. METHODS: We applied a systems approach to retrospectively analyse serological responses to Shigella across endemic and non-endemic populations. We profiled serum samples collected from 44 individuals from the USA without previous exposure to Shigella and who were experimentally challenged with Shigella sonnei (non-endemic setting), and serum samples collected from 55 Peruvian army recruits (endemic setting). In the endemic setting, a subset of 37 samples collected from individuals infected with culture-confirmed Shigella flexneri 2a were divided into two groups: susceptible, which included individuals infected within 90 days of entering the camp (n=29); or resistant, which included individuals infected later than 90 days after entering the camp (n=8). We analysed Shigella-specific antibody isotype, subclass, and Fc receptor binding profiles across IpaB, IpaC, IpaD, and lipopolysaccharide from S flexneri 2a, 3a, and 6, and S sonnei, and O-specific polysaccharide (OSP) from S flexneri 2a and 3a and S sonnei. We also evaluated antibody-mediated complement deposition and innate immune cell activation. The main outcome of interest was the detection of antibody markers and functionality associated with protection against shigellosis in a high-burden endemic setting. FINDINGS: Adults with endemic exposure to Shigella possessed broad and functional antibody responses across polysaccharide, glycolipid, and protein antigens compared with individuals from non-endemic regions. In a setting with high Shigella burden, elevated levels of OSP-specific Fcα receptor (FcαR) binding antibodies were associated with resistance to shigellosis, whereas total OSP-specific IgA was not, suggesting a potentially unique functionality. OSP-specific FcαR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation, and production of reactive oxygen species. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcαR and antibody-mediated activation of neutrophils and monocytes. INTERPRETATION: Our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against Shigella infection in a high-burden setting. These findings will assist in the development and evaluation of Shigella vaccines. FUNDING: US National Institutes of Health.

Exploring Natural Immune Responses to Shigella Exposure Using Multiplex Bead Assays on Dried Blood Spots in High-Burden Countries: Protocol From a Multisite Diarrhea Surveillance Study.

Background: Molecular diagnostics on human fecal samples have identified a larger burden of shigellosis than previously appreciated by culture. Evidence of fold changes in immunoglobulin G (IgG) to conserved and type-specific Shigella antigens could be used to validate the molecular assignment of type-specific Shigella as the etiology of acute diarrhea and support polymerase chain reaction (PCR)-based microbiologic end points for vaccine trials. Methods: We will test dried blood spots collected at enrollment and 4 weeks later using bead-based immunoassays for IgG to invasion plasmid antigen B and type-specific lipopolysaccharide O-antigen for Shigella flexneri 1b, 2a, 3a, and 6 and Shigella sonnei in Shigella-positive cases and age-, site-, and season-matched test-negative controls from all sites in the Enterics for Global Health (EFGH) Shigella surveillance study. Fold antibody responses will be compared between culture-positive, culture-negative but PCR-attributable, and PCR-positive but not attributable cases and test-negative controls. Age- and site-specific seroprevalence distributions will be identified, and the association between baseline antibodies and Shigella attribution will be estimated. Conclusions: The integration of these assays into the EFGH study will help support PCR-based attribution of acute diarrhea to type-specific Shigella, describe the baseline seroprevalence of conserved and type-specific Shigella antibodies, and support correlates of protection for immunity to Shigella diarrhea. These insights can help support the development and evaluation of Shigella vaccine candidates.

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.

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.

Bioactivity and efficacy of a hyperimmune bovine colostrum product- Travelan, against shigellosis in a non-Human primate model (Macaca mulatta).

Infectious diarrhea is a World Health Organization public health priority area due to the lack of effective vaccines and an accelerating global antimicrobial resistance crisis. New strategies are urgently needed such as immunoprophylactic for prevention of diarrheal diseases. Hyperimmune bovine colostrum (HBC) is an established and effective prophylactic for infectious diarrhea. The commercial HBC product, Travelan® (Immuron Ltd, Australia) targets multiple strains of enterotoxigenic Escherichia coli (ETEC) is highly effective in preventing diarrhea in human clinical studies. Although Travelan® targets ETEC, preliminary studies suggested cross-reactivity with other Gram-negative enteric pathogens including Shigella and Salmonella species. For this study we selected an invasive diarrheal/dysentery-causing enteric pathogen, Shigella, to evaluate the effectiveness of Travelan®, both in vitro and in vivo. Here we demonstrate broad cross-reactivity of Travelan® with all four Shigella spp. (S. flexneri, S. sonnei, S. dysenteriae and S. boydii) and important virulence factor Shigella antigens. Naïve juvenile rhesus macaques (NJRM) were randomized, 8 dosed with Travelan® and 4 with a placebo intragastrically twice daily over 6 days. All NJRM were challenged with S. flexneri 2a strain 2457T on the 4th day of treatment and monitored for diarrheal symptoms. All placebo-treated NJRM displayed acute dysentery symptoms within 24-36 hours of challenge. Two Travelan®-treated NJRM displayed dysentery symptoms and six animals remained healthy and symptom-free post challenge; resulting in 75% efficacy of prevention of shigellosis (p = 0.014). These results strongly indicate that Travelan® is functionally cross-reactive and an effective prophylactic for shigellosis. This has positive implications for the prophylactic use of Travelan® for protection against both ETEC and Shigella spp. diarrheal infections. Future refinement and expansion of pathogens recognized by HBC including Travelan® could revolutionize current management of gastrointestinal infections and outbreaks in travelers' including military, peacekeepers, humanitarian workers and in populations living in endemic regions of the world.

A broadly immunogenic polyvalent Shigella multiepitope fusion antigen protein protects against Shigella sonnei and Shigella flexneri lethal pulmonary challenges in mice.

There are no licensed vaccines for Shigella, a leading cause of children's diarrhea and a common etiology of travelers' diarrhea. To develop a cross-protective Shigella vaccine, in this study, we constructed a polyvalent protein immunogen to present conserved immunodominant epitopes of Shigella invasion plasmid antigens B (IpaB) and D (IpaD), VirG, GuaB, and Shiga toxins on backbone protein IpaD, by applying an epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform, examined protein (Shigella MEFA) broad immunogenicity, and evaluated antibody function against Shigella invasion and Shiga toxin cytotoxicity but also protection against Shigella lethal challenge. Mice intramuscularly immunized with Shigella MEFA protein developed IgG responses to IpaB, IpaD, VirG, GuaB, and Shiga toxins 1 and 2; mouse sera significantly reduced invasion of Shigella sonnei, Shigella flexneri serotype 2a, 3a, or 6, Shigella boydii, and Shigella dysenteriae type 1 and neutralized cytotoxicity of Shiga toxins of Shigella and Shiga toxin-producing Escherichia coli in vitro. Moreover, mice intranasally immunized with Shigella MEFA protein (adjuvanted with dmLT) developed antigen-specific serum IgG, lung IgG and IgA, and fecal IgA antibodies, and survived from lethal pulmonary challenge with S. sonnei or S. flexneri serotype 2a, 3a, or 6. In contrast, the control mice died, became unresponsive, or lost 20% of body weight in 48 h. These results indicated that this Shigella MEFA protein is broadly immunogenic, induces broadly functional antibodies, and cross-protects against lethal pulmonary challenges with S. sonnei or S. flexneri serotypes, suggesting a potential application of this polyvalent MEFA protein in Shigella vaccine development.

GMP manufacture of Shigella flexneri 2a Artificial Invaplex (InvaplexAR) and evaluation in a Phase 1 Open-label, dose escalating study administered intranasally to healthy, adult volunteers.

Shigella species cause severe disease among travelers to, and children living in, endemic countries. Although significant efforts have been made to improve sanitation, increased antibiotic resistance and other factors suggest an effective vaccine is a critical need. Artificial Invaplex (InvaplexAR) is a subunit vaccine approach complexing Shigella LPS with invasion plasmid antigens. In pre-clinical studies, the InvaplexAR vaccine demonstrated increased immunogenicity as compared to the first generation product and was subsequently manufactured under cGMP for clinical testing in a first-in-human Phase 1 study. The primary objective of this study was the safety of S. flexneri 2a InvaplexAR given by intranasal (IN) immunization (without adjuvant) in a single-center, open-label, dose-escalating Phase 1 trial and secondarily to assess immunogenicity to identify a dose of InvaplexAR for subsequent clinical evaluations. Subjects received three IN immunizations of InvaplexAR, two weeks apart, in increasing dose cohorts (10 µg, 50 µg, 250 µg, and 500 µg). Adverse events were monitored using symptom surveillance, memory aids, and targeted physical exams. Samples were collected throughout the study to investigate vaccine-induced systemic and mucosal immune responses. There were no adverse events that met vaccination-stopping criteria. The majority (96%) of vaccine-related adverse events were mild in severity (most commonly nasal congestion, rhinorrhea, and post-nasal drip). Vaccination with InvaplexAR induced anti-LPS serum IgG responses and anti-Invaplex IgA and IgG antibody secreting cell (ASC) responses at vaccine doses ≥250 µg. Additionally, mucosal immune responses and functional antibody responses were seen from the serum bactericidal assay measurements. Notably, the responder rates and the kinetics of ASCs and antibody lymphocyte secretion (ALS) were similar, suggesting that either assay may be employed to identify IgG and IgA secreting cells. Further studies with InvaplexAR will evaluate alternative immunization routes, vaccination schedules and formulations to further optimize immunogenicity. (Clinical Trial Registry Number NCT02445963).

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.