Protein-based Nanoparticle Vaccine Approaches Against Infectious Diseases.

The field of vaccine development has seen an increase in the number of rationally designed technologies that increase effectiveness against vaccine-resistant pathogens, while not compromising safety. Yet, there is still an urgent need to expand and further understand these platforms against complex pathogens that often evade protective responses. Nanoscale platforms have been at the center of new studies, especially in the wake of the coronavirus disease 2019 (COVID-19), with the aim of deploying safe and effective vaccines in a short time period. The intrinsic properties of protein-based nanoparticles, such as biocompatibility, flexible physicochemical characteristics, and variety have made them an attractive platform against different infectious disease agents. In the past decade, several studies have tested both lumazine synthase-, ferritin-, and albumin-based nanoplatforms against a wide range of complex pathogens in pre-clinical studies. Owed to their success in pre-clinical studies, several studies are undergoing human clinical trials or are near an initial phase. In this review we highlight the different protein-based platforms, mechanisms of synthesis, and effectiveness of these over the past decade. In addition, some challenges, and future directions to increase their effectiveness are also highlighted. Taken together, protein-based nanoscaffolds have proven to be an effective means to design rationally designed vaccines, especially against complex pathogens and emerging infectious diseases.

Dual RNA-seq reveals a type 6 secretion system-dependent blockage of TNF-α signaling and BicA as a Burkholderia pseudomallei virulence factor important during gastrointestinal infection.

Melioidosis is a disease caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm), commonly found in soil and water of endemic areas. Naturally acquired human melioidosis infections can result from either exposure through percutaneous inoculation, inhalation, or ingestion of soil-contaminated food or water. Our prior studies recognized Bpm as an effective enteric pathogen, capable of establishing acute or chronic gastrointestinal infections following oral inoculation. However, the specific mechanisms and virulence factors involved in the pathogenesis of Bpm during intestinal infection are unknown. In our current study, we standardized an in vitro intestinal infection model using primary intestinal epithelial cells (IECs) and demonstrated that Bpm requires a functional T6SS for full virulence. Further, we performed dual RNA-seq analysis on Bpm-infected IECs to evaluate differentially expressed host and bacterial genes in the presence or absence of a T6SS. Our results showed a dysregulation in the TNF-α signaling via NF-κB pathway in the absence of the T6SS, with some of the genes involved in inflammatory processes and cell death also affected. Analysis of the bacterial transcriptome identified virulence factors and regulatory proteins playing a role during infection, with association to the T6SS. By using a Bpm transposon mutant library and isogenic mutants, we showed that deletion of the bicA gene, encoding a putative T3SS/T6SS regulator, ablated intracellular survival and plaque formation by Bpm and impacted survival and virulence when using murine models of acute and chronic gastrointestinal infection. Overall, these results highlight the importance of the type 6 secretion system in the gastrointestinal pathogenesis of Bpm.

Optimization of Multivalent Gold Nanoparticle Vaccines Eliciting Humoral and Cellular Immunity in an In Vivo Model of Enterohemorrhagic Escherichia coli O157:H7 Colonization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 remains a pathogen of significance and high consequence around the world. This outcome is due in part to the high economic impact associated with massive, contaminated product recalls, prevalence of the pathogen in carrier reservoirs, disease sequelae, and mortality associated with several outbreaks worldwide. Furthermore, the contraindication of antibiotic use for the treatment of EHEC-related infections makes this pathogen a primary candidate for the development of effective prophylactic vaccines. However, no vaccines are approved for human use, and many have failed to provide a high degree of efficacy or broad protection, thereby opening an avenue for the use of new technologies to produce a safe, effective, and protective vaccine. Building on our previous studies using reverse vaccinology-predicted antigens, we refine a formulation, evaluate new immunogenic antigens, and further expand our understanding about the mechanism of EHEC vaccine-mediated protection. In the current study, we exploit the use of the nanotechnology platform based on gold nanoparticles (AuNP), which can act as a scaffold for the delivery of various antigens. Our results demonstrate that a refined vaccine formulation incorporating EHEC antigen LomW, EscC, LpfA1, or LpfA2 and delivered using AuNPs can elicit robust antigen-specific cellular and humoral responses associated with reduced EHEC colonization in vivo. Furthermore, our in vitro mechanistic studies further support that antibody-mediated protection is primarily driven by inhibition of bacterial adherence onto intestinal epithelial cells and by promotion of macrophage uptake and killing. IMPORTANCE Enterohemorrhagic E. coli O157:H7 remains an important human pathogen that does not have an effective and safe vaccine available. We have made outstanding progress in the identification of novel protective antigens that have been incorporated into the gold nanoparticle platform and used as vaccines. In this study, we have refined our vaccine formulations to incorporate multiple antigens and further define the mechanism of antibody-mediated protection, including one vaccine that promotes macrophage uptake. We further define the cell-mediated responses elicited at the mucosal surface by our nanovaccine formulations, another key immune mechanism linked to protection.

Multicomponent Gold-Linked Glycoconjugate Vaccine Elicits Antigen-Specific Humoral and Mixed TH1-TH17 Immunity, Correlated with Increased Protection against Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal disease with a high mortality rate. The intrinsic resistance to commonly used antibiotics combined with the complex bacterial life cycle has hampered the development of preventive and therapeutic interventions and vaccines. Furthermore, the need of humoral and cell-mediated immunity in protection against B. pseudomallei has complicated the development of effective vaccines. Antigen delivery vaccine platforms that promote humoral and cellular responses while maintaining a safe profile are a roadblock to developing subunit vaccines against intracellular pathogens. Gold nanoparticles (AuNPs) were used for the delivery of multicomponent antigens with the goal of inducing vaccine-mediated immunity, promoting protection against melioidosis disease. Different nanoglycoconjugates using predicted immunogenic protein candidates, Hcp1, FlgL, OpcP, OpcP1, OmpW, and hemagglutinin, were covalently coupled to AuNPs, together with the lipopolysaccharide (LPS) from Burkholderia thailandensis, which acted as an additional antigen. Animals immunized with individually coupled (AuNP-protein-LPS) formulations containing OpcP or OpcP1, together with CpG as an adjuvant, showed a significant increase in protection, whereas a nanovaccine combination (AuNP-Combo2-LPS) showed significant and complete protection against a lethal intranasal B. pseudomallei challenge. Animals immunized with AuNP-Combo2-LPS showed robust humoral antigen-specific (IgG and IgA) responses with higher IgG2c titer, indicating a TH1-skewed response and promotion of macrophage uptake. In addition, immunization with the nanovaccine combination resulted in a mixed antigen-specific TH1-TH17 cytokine profile after immunization. This study provides the basis for an elegant and refined multicomponent glycoconjugate vaccine formulation capable of eliciting both humoral and cell-mediated responses against lethal B. pseudomallei challenge. IMPORTANCE Melioidosis is a complex human disease associated with a wide range of complications caused by the Gram-negative bacillus Burkholderia pseudomallei. The global burden of melioidosis is estimated to have 165,000 cases per year and 89,000 fatal outcomes. The endemicity of B. pseudomallei includes a wide range of tropical regions in Asia, Africa, Latin America, and Australia. Therefore, a viable alternative to prevent human infections is the development of an effective vaccine; however, no approved vaccine for human use is available. This study provides a vaccine strategy against B. pseudomallei and an immune-stimulatory platform to induce strong humoral and T-cell-mediated immunity.

Burkholderia pseudomallei as an Enteric Pathogen: Identification of Virulence Factors Mediating Gastrointestinal Infection.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, B. pseudomallei poses a significant health risk, especially in regions of endemicity, where treatment requires prolonged antibiotic therapy. Even though the respiratory and percutaneous routes are well documented and considered the main ways to acquire the pathogen, the gastrointestinal tract is believed to be an underreported and underrecognized route of infection. In the present study, we describe the development of in vitro and in vivo models to study B. pseudomallei gastrointestinal infection. Further, we report that the type 6 secretion system (T6SS) and type 1 fimbriae are important virulence factors required for gastrointestinal infection. Using a human intestinal epithelial cell line and mouse primary intestinal epithelial cells (IECs), we demonstrated that B. pseudomallei adheres, invades, and forms multinucleated giant cells, ultimately leading to cell toxicity. We demonstrated that mannose-sensitive type 1 fimbria is involved in the initial adherence of B. pseudomallei to IECs, although the impact on full virulence was limited. Finally, we also showed that B. pseudomallei requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Overall, we showed that B. pseudomallei is an enteric pathogen and that type 1 fimbria is important for B. pseudomallei intestinal adherence, and we identify a new role for T6SS as a key virulence factor in gastrointestinal infection. These studies highlight the importance of gastrointestinal melioidosis as an understudied route of infection and open a new avenue for the pathogenesis of B. pseudomallei.

Multicomponent gold nano-glycoconjugate as a highly immunogenic and protective platform against Burkholderia mallei.

Burkholderia mallei (Bm) is a facultative intracellular pathogen and the etiological agent of glanders, a highly infectious zoonotic disease occurring in equines and humans. The intrinsic resistance to antibiotics, lack of specific therapy, high mortality, and history as a biothreat agent, prompt the need of a safe and effective vaccine. However, the limited knowledge of protective Bm-specific antigens has hampered the development of a vaccine. Further, the use of antigen-delivery systems that enhance antigen immunogenicity and elicit robust antigen-specific immune responses has been limited and could improve vaccines against Bm. Nanovaccines, in particular gold nanoparticles (AuNPs), have been investigated as a strategy to broaden the repertoire of vaccine-mediated immunity and as a tool to produce multivalent vaccines. To synthesize a nano-glycoconjugate vaccine, six predicted highly immunogenic antigens identified by a genome-wide bio- and immuno-informatic analysis were purified and coupled to AuNPs along with lipopolysaccharide (LPS) from B. thailandensis. Mice immunized intranasally with individual AuNP-protein-LPS conjugates, showed variable degrees of protection against intranasal Bm infection, while an optimized combination formulation (containing protein antigens OmpW, OpcP, and Hemagglutinin, along with LPS) showed complete protection against lethality in a mouse model of inhalational glanders. Animals immunized with different nano-glycoconjugates showed robust antigen-specific antibody responses. Moreover, serum from animals immunized with the optimized nano-glycoconjugate formulation showed sustained antibody responses with increased serum-mediated inhibition of adherence and opsonophagocytic activity in vitro. This study provides the basis for the rational design and construction of a multicomponent vaccine platform against Bm.

Curcumin Blocks Cytotoxicity of Enteroaggregative and Enteropathogenic Escherichia coli by Blocking Pet and EspC Proteolytic Release From Bacterial Outer Membrane.

Pet and EspC are toxins secreted by enteroaggregative (EAEC) and enteropathogenic (EPEC) diarrheagenic Escherichia coli pathotypes, respectively. Both toxins are members of the Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) family. Pet and EspC are important virulence factors that produce cytotoxic and enterotoxic effects on enterocytes. Here, we evaluated the effect of curcumin, a polyphenolic compound obtained from the rhizomes of Curcuma longa L. (Zingiberaceae) on the secretion and cytotoxic effects of Pet and EspC proteins. We found that curcumin prevents Pet and EspC secretion without affecting bacterial growth or the expression of pet and espC. Our results show that curcumin affects the release of these SPATEs from the translocation domain, thereby affecting the pathogenesis of EAEC and EPEC. Curcumin-treated EAEC and EPEC did not induce significant cell damage like the ability to disrupt the actin cytoskeleton, without affecting their characteristic adherence patterns on epithelial cells. A molecular model of docking predicted that curcumin interacts with the determinant residues Asp1018-Asp1019 and Asp1029-Asp1030 of the translocation domain required for the release of Pet and EspC, respectively. Consequently, curcumin blocks Pet and EspC cytotoxicity on epithelial cells by preventing their release from the outer membrane.

Emerging role of biologics for the treatment of melioidosis and glanders.

Introduction: Two important pathogenic species within the genus Burkholderia, namely Burkholderia pseudomallei (Bpm) and Burkholderia mallei (Bm), are the causative agents of the life-threatening diseases melioidosis and glanders, respectively. Due to their high mortality rate and potential for aerosolization, they have gained interest as potential biothreat agents and are classified as Tier 1 Select Agents.Areas covered: The manuscript provides an overview of the literature covering the efforts taken in the last 10 years to develop new therapeutics measures against both Bpm and Bm, with attention on novel therapeutic agents.Expert Opinion: As a result of the complicated antibiotic regimens necessary to treat these infections, development of novel therapeutics is needed to treat both diseases. In recent years, the understanding of the pathogenesis of Burkholderia has improved significantly and so have the efforts to develop novel therapeutic agents with high efficacy, either alone, or in combination with conventional antibiotics.

Development of a Gold Nanoparticle Vaccine against Enterohemorrhagic Escherichia coli O157:H7.

Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichiacoli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens.IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models.

Melioidosis in Mexico, Central America, and the Caribbean.

Burkholderia pseudomallei is the causative agent of melioidosis, an endemic disease in tropical areas around the world. Cumulative human cases have demonstrated that melioidosis is prevalent and increasingly recognized in the American continent. Even though the first reports of melioidosis in Mexico, Central America, and the Caribbean Islands date back to the late 1940s, the potential of the disease as a public health concern in the region has not been fully appreciated. Unfortunately, recent studies predicting the global distribution of the disease and the demonstration of melioidosis endemicity in Puerto Rico have not increased recognition of the disease by health professionals in this region. Furthermore, a lack of both diagnostic capacity and awareness of the disease has resulted in a limited number of studies that have attempted to accurately determine its prevalence and geographical distribution. In this review, a summary of reported cases in the countries of this region are presented, as well as recommendations to increase the diagnosis and awareness of the disease as an important public health problem in Mexico, Central America, and the Caribbean islands.