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.

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.

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 the Shigella flexneri 2a, 3a, 6, and S. sonnei artificial Invaplex (InvaplexAR) vaccines.

The Shigella artificial invasin complex (InvaplexAR) vaccine is a subunit approach that effectively induces robust immunogenicity directed to serotype-specific lipopolysaccharide and the broadly conserved IpaB and IpaC proteins. One advantage of the vaccine approach is the ability to adjust the constituents to address suboptimal immunogenicity and to change the Shigella serotype targeted by the vaccine. As the vaccine moves through the product development pipeline, substantial modifications have been made to address manufacturing feasibility, acceptability to regulatory authorities, and developing immunogenic and effective products for an expanded list of Shigella serotypes. Modifications of the recombinant clones used to express affinity tag-free proteins using well-established purification methods, changes to detergents utilized in the assembly process, and in vitro and in vivo evaluation of different Invaplex formulations have led to the establishment of a scalable, reproducible manufacturing process and enhanced immunogenicity of Invaplex products designed to protect against four of the most predominant Shigella serotypes responsible for global morbidity and mortality. These adjustments and improvements provide the pathway for the manufacture and clinical testing of a multivalent Invaplex vaccine. IMPORTANCE Shigella species are a major global health concern that cause severe diarrhea and dysentery in children and travelers to endemic areas of the world. Despite significant advancements in access to clean water, the increases in antimicrobial resistance and the risk of post-infection sequelae, including cognitive and physical stunting in children, highlight the urgent need for an efficacious vaccine. One promising vaccine approach, artificial Invaplex, delivers key antigens recognized by the immune system during infection, which results in increased resistance to re-infection. The work presented here describes novel modifications to a previously described vaccine approach resulting in improved methods for manufacturing and regulatory approvals, expansion of the breadth of coverage to all major Shigella serotypes, and an increase in the potency of artificial Invaplex.

Shigella-Controlled Human Infection Models: Current and Future Perspectives.

Shigella-controlled human infection models (CHIMs) are an invaluable tool utilized by the vaccine community to combat one of the leading global causes of infectious diarrhea, which affects infants, children and adults regardless of socioeconomic status. The impact of shigellosis disproportionately affects children in low- and middle-income countries (LMICs) resulting in cognitive and physical stunting, perpetuating a cycle that must be halted. Shigella-CHIMs not only facilitate the early evaluation of enteric countermeasures and up-selection of the most promising products but also provide insight into mechanisms of infection and immunity that are not possible utilizing animal models or in vitro systems. The greater understanding of shigellosis obtained in CHIMs builds and empowers the development of new generation solutions to global health issues which are unattainable in the conventional laboratory and clinical settings. Therefore, refining, mining and expansion of safe and reproducible infection models hold the potential to create effective means to end diarrheal disease and associated co-morbidities associated with Shigella infection.

From Concept to Clinical Product: A Brief History of the Novel Shigella Invaplex Vaccine's Refinement and Evolution.

The Shigella invasin complex or Invaplex vaccine is a unique subunit approach to generate a protective immune response. Invaplex is a large, macromolecular complex consisting of the major Shigella antigens: lipopolysaccharide (LPS) and the invasion plasmid antigen (Ipa) proteins B and C. Over the past several decades, the vaccine has progressed from initial observations through pre-clinical studies to cGMP manufacture and clinical evaluations. The Invaplex product maintains unique biological properties associated with the invasiveness of virulent shigellae and also presents both serotype-specific epitopes, as well as highly conserved invasin protein epitopes, to the immunized host. The vaccine product has evolved from a native product isolated from wild-type shigellae (native Invaplex) to a more defined vaccine produced from purified LPS and recombinant IpaB and IpaC (artificial Invaplex). Each successive "generation" of the vaccine is derived from earlier versions, resulting in improved immunogenicity, homogeneity and effectiveness. The current vaccine, detoxified artificial Invaplex (InvaplexAR-Detox), was developed for parenteral administration by incorporating LPS with under-acylated lipid A. InvaplexAR-Detox has demonstrated an excellent safety and immunogenicity profile in initial clinical studies and is advancing toward evaluations in the target populations of children and travelers to endemic countries.

Efficacy, safety, and immunogenicity of the Shigella sonnei 1790GAHB GMMA candidate vaccine: Results from a phase 2b randomized, placebo-controlled challenge study in adults.

BACKGROUND: Shigellosis accounts for substantial morbidity and mortality worldwide and is the second most common cause of moderate and severe diarrhoea in children. METHODS: This phase 2b study (NCT03527173), conducted between August 2018 and November 2019, evaluated vaccine efficacy (VE), safety, and immunogenicity of a Shigella sonnei GMMA candidate vaccine (1790GAHB) in adults, using a S. sonnei 53 G controlled human infection model. Participants (randomized 1:1) received two doses of 1790GAHB or placebo (GAHB-Placebo), at day (D) 1 and D29, and an oral challenge of S. sonnei 53 G at D57. VE was evaluated using several endpoints, reflecting different case definitions of shigellosis. For the primary endpoint, the success criterion was a lower limit of the 90% confidence interval >0. FINDINGS: Thirty-six and 35 participants received 1790GAHB or placebo, respectively; 33 and 29 were challenged, 15 and 12 developed shigellosis. VE was not demonstrated for any endpoint. Adverse events were more frequent in 1790GAHB versus placebo recipients post-vaccination. Anti-S. sonnei lipopolysaccharide (LPS) IgG responses increased at D29 and remained stable through D57 in group 1790GAHB; no increase was shown in placebo recipients. INTERPRETATION: 1790GAHB had an acceptable safety profile and induced anti-LPS IgG responses but did not demonstrate clinical efficacy against shigellosis. Baseline/pre-challenge antibody levels were higher in participants who did not develop shigellosis post-challenge, suggesting a role of anti-LPS IgG antibodies in clinical protection, although not fully elucidated in this study. For further vaccine development an increased S. sonnei O-antigen content is likely needed to enhance anti-LPS immune responses. FUNDING: GlaxoSmithKline Biologicals SA, Bill and Melinda Gates Foundation.

Shigella-Specific Immune Profiles Induced after Parenteral Immunization or Oral Challenge with Either Shigella flexneri 2a or Shigella sonnei.

Shigella spp. are a leading cause of diarrhea-associated global morbidity and mortality. Development and widespread implementation of an efficacious vaccine remain the best option to reduce Shigella-specific morbidity. Unfortunately, the lack of a well-defined correlate of protection for shigellosis continues to hinder vaccine development efforts. Shigella controlled human infection models (CHIM) are often used in the early stages of vaccine development to provide preliminary estimates of vaccine efficacy; however, CHIMs also provide the opportunity to conduct in-depth immune response characterizations pre- and postvaccination or pre- and postinfection. In the current study, principal-component analyses were used to examine immune response data from two recent Shigella CHIMs in order to characterize immune response profiles associated with parenteral immunization, oral challenge with Shigella flexneri 2a, or oral challenge with Shigella sonnei. Although parenteral immunization induced an immune profile characterized by robust systemic antibody responses, it also included mucosal responses. Interestingly, oral challenge with S. flexneri 2a induced a distinctively different profile compared to S. sonnei, characterized by a relatively balanced systemic and mucosal response. In contrast, S. sonnei induced robust increases in mucosal antibodies with no differences in systemic responses across shigellosis outcomes postchallenge. Furthermore, S. flexneri 2a challenge induced significantly higher levels of intestinal inflammation compared to S. sonnei, suggesting that both serotypes may also differ in how they trigger induction and activation of innate immunity. These findings could have important implications for Shigella vaccine development as protective immune mechanisms may differ across Shigella serotypes. IMPORTANCE Although immune correlates of protection have yet to be defined for shigellosis, prior studies have demonstrated that Shigella infection provides protection against reinfection in a serotype-specific manner. Therefore, it is likely that subjects with moderate to severe disease post-oral challenge would be protected from a homologous rechallenge, and investigating immune responses in these subjects may help identify immune markers associated with the development of protective immunity. This is the first study to describe distinct innate and adaptive immune profiles post-oral challenge with two different Shigella serotypes. Analyses conducted here provide essential insights into the potential of different immune mechanisms required to elicit protective immunity, depending on the Shigella serotype. Such differences could have significant impacts on vaccine design and development within the Shigella field and should be further investigated across multiple Shigella serotypes.

Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlate of protection.

BACKGROUND: Shigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis. METHODS: In this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed. FINDINGS: Sixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002). INTERPRETATION: The Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) FUNDING: Funding for this study was through a grant from the Wellcome Trust.

Immune response characterization in a human challenge study with a Shigella flexneri 2a bioconjugate vaccine.

BACKGROUND: Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. METHODS: Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. FINDINGS: Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. INTERPRETATION: Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. FUNDING: Funding for this study was provided through a Wellcome Trust grant.

Establishment of a Controlled Human Infection Model with a Lyophilized Strain of Shigella sonnei 53G.

Controlled human infection models (CHIMs) are useful for vaccine development. To improve on existing models, we developed a CHIM using a lyophilized preparation of Shigella sonnei strain 53G produced using current good manufacturing practice (cGMP). Healthy adults were enrolled in an open-label dose-ranging study. Following administration of a dose of rehydrated S. sonnei strain 53G, subjects were monitored for development of disease. The first cohort received 500 CFU of 53G, and dosing of subsequent cohorts was based on results from the previous cohort. Subjects were administered ciprofloxacin on day 5 and discharged home on day 8. Subjects returned as outpatients for clinical checks and sample collection. Attack rates increased as the dose of S. sonnei was increased. Among those receiving the highest dose (1,760 CFU), 70% developed moderate to severe diarrhea, 50% had dysentery, and 40% had fever. Antilipopolysaccharide responses were observed across all cohorts. An S. sonnei CHIM using a lyophilized lot of strain 53G was established. A dose in the range of 1,500 to 2,000 CFU of 53G was selected as the dose for future challenge studies using this product. This model will enable direct comparison of study results between institutions and ensure better consistency over time in the challenge inoculum.IMPORTANCE Controlled human infection models (CHIMs) are invaluable tools utilized to understand the human response to infection, potentially leading to protective immune mechanisms and allowing efficacy testing of enteric countermeasures, including vaccines, antibiotics, and other products. The development of an improved Shigella CHIM for both Shigella sonnei and Shigella flexneri is consistent with international efforts, supported by international donors and the World Health Organization, focused on standardizing Shigella CHIMs and using them to accelerate Shigella vaccine development. The use of lyophilized Shigella challenge strains rather than plate-grown inoculum preparations is considered an important step forward in the standardization process. Furthermore, the results of studies such as this justify the development of lyophilized preparations for additional epidemiologically important S. flexneri serotypes, including S. flexneri 3a and S. flexneri 6.

Immune Response Characterization after Controlled Infection with Lyophilized Shigella sonnei 53G.

Shigella is a major cause of moderate to severe diarrhea largely affecting children (<5 years old) living in low- and middle-income countries. Several vaccine candidates are in development, and controlled human infection models (CHIMs) can be useful tools to provide an early assessment of vaccine efficacy and potentially support licensure. A lyophilized strain of S. sonnei 53G was manufactured and evaluated to establish a dose that safely and reproducibly induced a ≥60% attack rate. Samples were collected pre- and postchallenge to assess intestinal inflammatory responses, antigen-specific serum and mucosal antibody responses, functional antibody responses, and memory B cell responses. Infection with S. sonnei 53G induced a robust intestinal inflammatory response as well as antigen-specific antibodies in serum and mucosal secretions and antigen-specific IgA- and IgG-secreting B cells positive for the α4ß7 gut-homing marker. There was no association between clinical disease outcomes and systemic or functional antibody responses postchallenge; however, higher lipopolysaccharide (LPS)-specific serum IgA- and IgA-secreting memory B cell responses were associated with a reduced risk of disease postchallenge. This study provides unique insights into the immune responses pre- and postinfection with S. sonnei 53G in a CHIM, which could help guide the rational design of future vaccines to induce protective immune responses more analogous to those triggered by infection.IMPORTANCE Correlate(s) of immunity have yet to be defined for shigellosis. As previous disease protects against subsequent infection in a serotype-specific manner, investigating immune response profiles pre- and postinfection provides an opportunity to identify immune markers potentially associated with the development of protective immunity and/or with a reduced risk of developing shigellosis postchallenge. This study is the first to report such an extensive characterization of the immune response after challenge with S. sonnei 53G. Results demonstrate an association of progression to shigellosis with robust intestinal inflammatory and mucosal gut-homing responses. An important finding in this study was the association of elevated Shigella LPS-specific serum IgA and memory B cell IgA responses at baseline with reduced risk of disease. The increased baseline IgA responses may contribute to the lack of dose response observed in the study and suggests that IgA responses should be further investigated as potential correlates of immunity.

Consensus Report on Shigella Controlled Human Infection Model: Immunological Assays.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

Assembly, Biochemical Characterization, Immunogenicity, Adjuvanticity, and Efficacy of Shigella Artificial Invaplex.

The native Invaplex (InvaplexNAT) vaccine and adjuvant is an ion exchange-purified product derived from the water extract of virulent Shigella species. The key component of InvaplexNAT is a high-molecular-mass complex (HMMC) consisting of the Shigella lipopolysaccharide (LPS) and the invasin proteins IpaB and IpaC. To improve product purity and immunogenicity, artificial Invaplex (InvaplexAR) was developed using recombinant IpaB and IpaC proteins and purified Shigella LPS to assemble an HMMC consisting of all three components. Characterization of InvaplexAR by various methods demonstrated similar characteristics as the previously reported HMMC in InvaplexNAT. The well-defined InvaplexAR vaccine consistently contained greater quantities of IpaB, IpaC, and LPS than InvaplexNAT. InvaplexAR and InvaplexNAT immunogenicities were compared in mouse and guinea pig dose escalation studies. In both models, immunization induced antibody responses specific for InvaplexNAT and LPS while InvaplexAR induced markedly higher anti-IpaB and -IpaC serum IgG and IgA endpoint titers. In the murine model, homologous protection was achieved with 10-fold less InvaplexAR than InvaplexNAT and mice receiving InvaplexAR lost significantly less weight than mice receiving the same amount of InvaplexNAT. Moreover, mice immunized with InvaplexAR were protected from challenge with both homologous and heterologous Shigella serotypes. Guinea pigs receiving approximately 5-fold less InvaplexAR compared to cohorts immunized with InvaplexNAT were protected from ocular challenge. Furthermore, adjuvanticity previously attributed to InvaplexNAT was retained with InvaplexAR. The second-generation Shigella Invaplex vaccine, InvaplexAR, offers significant advantages over InvaplexNAT in reproducibility, flexible yet defined composition, immunogenicity, and protective efficacy. IMPORTANCEShigella species are bacteria that cause severe diarrheal disease worldwide, primarily in young children. Treatment of shigellosis includes oral fluids and antibiotics, but the high burden of disease, increasing prevalence of antibiotic resistance, and long-term health consequences clearly warrant the development of an effective vaccine. One Shigella vaccine under development is termed the invasin complex or Invaplex and is designed to drive an immune response to specific antigens of the bacteria in an effort to protect an individual from infection. The work presented here describes the production and evaluation of a new generation of Invaplex. The improved vaccine stimulates the production of antibodies in immunized mice and guinea pigs and protects these animals from Shigella infection. The next step in the product's development will be to test the safety and immune response induced in humans immunized with Invaplex.

Improving chances for successful clinical outcomes with better preclinical models.

In order to avoid expensive clinical failures, better and more predictive animal models of vaccine efficacy are needed to screen Shigella and ETEC vaccine candidates for protective efficacy. The 2016 Vaccines Against Shigella and ETEC (VASE) Conference included a workshop focused on the strengths and weaknesses of current models, particularly in terms of the correlation to vaccine efficacy in human clinical trials. Workshop presenters shared information on existing preclinical animal models for assessing the immunogenicity and protective efficacy of Shigella and ETEC vaccines. The presentations were followed by a discussion about how to best utilize these models, how the models can be improved, and best practices for Shigella and ETEC vaccine developers. The workshop concluded with three major recommendations for the field: (1) develop better and more consistent reagents for animal studies and make them widely available, (2) prioritize harmonization of animal models and immunology assays, and (3) develop preclinical correlates of protection, which will be key in selecting the best vaccine candidates for further clinical development.

Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study.

Several candidate vaccines against Shigella spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered Shigella vaccine development over the past several decades. Bioconjugation technology, exploited here for an Shigella flexneri 2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel S. flexneri 2a bioconjugate vaccine made of the polysaccharide component of the S. flexneri 2a O-antigen, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 µg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant S. flexneri 2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against S. flexneri 2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to S. flexneri 2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.).