Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4′ phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules’ biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

Shigella spp. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against Shigella, and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 (S. sonnei) and WRSf2G12 (S. flexneri 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the Shigella backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling in vitro and dampened endotoxic effects in vivo. These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective Shigella vaccines.

Background: The quest for well-defined immunoadjuvants remains one of the highest priorities for the successful development of effective vaccines. Combination adjuvants, which are designed to integrate both the ability to activate a variety of immune mechanisms and synergistically improve the delivery of vaccine components, are well-positioned to address the unmet needs. The development of a preventive vaccine against hepatitis C virus (HCV)—a major public health concern—is a particular instance in which the choice of the immunoadjuvant is of utmost importance. Methods: We assembled a lipid A Toll-like receptor 4 (TLR4) agonist BECC438 and TLR7/8 agonist resiquimod (R848) on a polyphosphazene macromolecule (PCPP) to create a nanoscale immunoadjuvant-vaccine delivery system: PCPP-R+BECC438. This aqueous-based system was formulated with the HCV sE2 antigen, and the resulting vaccine candidate was evaluated in vivo for the ability to induce immune responses. Results: Co-assembly of adjuvants resulted in a visually clear aqueous system of nanoscale dimensions, monomodal size distribution, and entropy-driven interactions between components. Intramuscular immunization of mice with HCV sE2 antigen formulated in a polyphosphazene-based nano-system induced ten-fold higher IgG and IgG2a titers than the antigen adjuvanted with BECC438 alone. PCPP-R+BECC438 formulated HCV sE2 also produced statistically significant improvements in IgG2a/IgG1 ratio and more robust HCVpp neutralization ID₅₀ titers than control formulations. Conclusions: Polyphosphazene-assembled adjuvant nano-system promotes in vivo immune responses of enhanced quantity and quality of antibodies with increased potency of HCV neutralization.

Shigella spp. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against Shigella, and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 (S. sonnei) and WRSf2G12 (S. flexneri 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the Shigella backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling in vitro and dampened endotoxic effects in vivo. These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective Shigella vaccines.

Background: The quest for well-defined immunoadjuvants remains one of the highest priorities for the successful development of effective vaccines. Combination adjuvants, which are designed to integrate both the ability to activate a variety of immune mechanisms and synergistically improve delivery of vaccine components, are well-positioned to address the unmet needs. The development of preventive vaccine against hepatitis C virus (HCV) – a major public health concern, is a particular instance in which choice of the immunoadjuvant is of utmost importance. Methods: We assembled a lipid A Toll-like receptor 4 (TLR4) agonist BECC438 and TLR7/8 agonist resiquimod (R848) on a polyphosphazene macromolecule (PCPP) to create a nanoscale immunoadjuvant-vaccine delivery system: PCPP-R+BECC438. This aqueous-based system was formulated with HCV sE2 antigen, and the resulting vaccine candidate was evaluated in vivo for the ability to induce immune responses. Results: Co-assembly of adjuvants resulted in a visually clear aqueous system of nanoscale dimensions, monomodal size distribution and entropy driven interactions between components. Intramuscular immunization of mice with HCV sE2 antigen formulated in polyphosphazene-based nano-system induced ten-fold higher IgG and IgG2a titers than the antigen adjuvanted with BECC438 alone. PCPP-R+BECC438 formulated HCV sE2 also produced statistically significant improvements in IgG2a/IgG1 ratio and more robust HCVpp neutralization ID50 titers than control formulations. Conclusions: Polyphosphazene-assembled adjuvant nano-system promotes in vivo immune responses of enhanced quantity and quality of antibodies with increased potency of HCV neutralization.