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.

[Reactivity to antigens of the microbiome of the respiratory tract in patients with respiratory allergic diseases]. / Reactividad a antígenos del microbioma de vías respiratorias en pacientes con enfermedad alérgica respiratoria.

BACKGROUND: The prevalence of allergic diseases has increased worldwide. Recent studies have informed that the dysbiosis of some specific members of the human microbiota may enhance the allergic response of the respiratory tract. OBJECTIVE: To retrospectively explore the role of some microorganisms of the human microbiota on the skin reactivity and their effect on the chronicity of allergic respiratory diseases in humans. METHODS: A retrospective analysis of a 5-year database of patients with allergic respiratory tract disease. The frequency and magnitude of the reactivity to 38 different allergens was determined. RESULTS: Dermatophagoides pteronyssinus had the highest frequency of reactivity (93.7 %), followed by the bacterial allergen (a mixture of Staphylococcus aureus and Staphylococcus epidermidis) with a frequency of reactivity of 91.82 %; whereas Candida albicans had a frequency of reactivity of only 79.32 %. The frequency of reactivity to the pollen of native Mexican weeds was even lower ~79 %. CONCLUSION: The microorganisms of the microbiota that were analyzed in this study seem to have an influence on the development of respiratory allergic inflammation, associated with long-term colonization of the pharynx, nasal mucosa, and sinuses because of these microorganisms.

Antecedentes: La prevalencia de las enfermedades alérgicas ha aumentado en todo el mundo. En estudios recientes se ha informado que la disbiosis de algunos miembros específicos de la microbiota humana puede potenciar la respuesta alérgica de las vías respiratorias. Objetivo: Explorar retrospectivamente el papel de algunos microorganismos de la microbiota humana en la reactividad cutánea y su efecto sobre la cronicidad de las enfermedades alérgicas respiratorias en el humano. Métodos: Análisis retrospectivo de la base de datos de un periodo de cinco años de pacientes con enfermedad alérgica de las vías respiratorias. Se determinó la frecuencia y magnitud de la reactividad a 38 alérgenos diferentes. Resultados: La mayor frecuencia de reactividad la presentó Dermatophagoides pteronyssinus (93.7 %), al que le siguió una combinación bacteriana de Staphylococcus aureus-Staphylococcus epidermidis (91.82 %) y Candida albicans (79.32 %). La reactividad a alérgenos de polen de malezas nativas de México fue aun menor, aproximadamente de 79 %. Conclusión: Los microorganismos de la microbiota analizados en este estudio parecen tener una influencia en el desarrollo de la inflamación alérgica respiratoria, asociada a la colonización a largo plazo de la faringe, la mucosa nasal y los senos paranasales.

Classification of breast tumor models with a prototype microwave imaging system.

PURPOSE: The assessment of the size and shape of breast tumors is of utter importance to the correct diagnosis and staging of breast cancer. In this paper, we classify breast tumor models of varying sizes and shapes using signals collected with a monostatic ultra-wideband radar microwave imaging prototype system with machine learning algorithms specifically tailored to the collected data. METHODS: A database comprising 13 benign and 13 malignant tumor models with sizes between 13 and 40 mm was created using dielectrically representative tissue mimicking materials. These tumor models were placed inside two breast phantoms: a homogeneous breast phantom and a breast phantom with clusters of fibroglandular mimicking tissue, accounting for breast heterogeneity. The breast phantoms with tumors were imaged with a monostatic microwave imaging prototype system, over a 1-6 GHz frequency range. The classification of benign and malignant tumors embedded in the two breast phantoms was completed, and tumor classification was evaluated with Principal Component Analysis as a feature extraction method, and tuned Naïve Bayes (NB), decision trees (DT), and k-nearest neighbours (kNN) as classifiers. We further study which antenna positions are better placed to classify tumors, discuss the feature extraction method and optimize classification algorithms, by tuning their hyperparameters, to improve sensitivity, specificity and the receiver operating characteristic curve, while ensuring maximum generalization and avoiding overfitting and data contamination. We also added a realistic synthetic skin response to the collected signals and examined its global effect on classification of benign vs malignant tumors. RESULTS: In terms of global classification performance, kNN outperformed DT and NB machine learning classifiers, achieving a classification accuracy of 96.2% when classifying between benign and malignant tumor phantoms in a homogeneous breast phantom (both when the skin artifact is and is not considered). CONCLUSIONS: We experimentally classified tumor models as benign or malignant with a microwave imaging system, and we showed a methodology that can potentially assess the shape of breast tumors, which will give further insight into the correct diagnosis and staging of breast cancer.

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.

Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis.

BACKGROUND: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strains in the susceptible BALB/c mouse model. METHODOLOGY/PRINCIPAL FINDING: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. CONCLUSIONS/SIGNIFICANCE: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies.

Empatía y estudiantes de kinesiología / Empathy in students of Kinesiology

RESUMEN Introducción: la empatía es un concepto importante en la interacción entre el profesional kinesiólogo y el paciente. Objetivo: determinar los niveles de empatía en estudiantes de kinesiología en los factores curso y género de la Facultad de Ciencias de la Salud, Universidad de Atacama (Chile) en noviembre 2018. Métodos: estudio exploratorio, transversal. Se estudió una muestra de 191 estudiantes. Se empleó la escala de Empatía de Jefferson. Fueron estimadas la mediana, cuartil 1 y 3, diferencia intercuartílica e intervalo de confianza de la mediana. Se estudiaron las posibles diferencias en la empatía mediante pruebas no paramétricas (Mediana de Mood). Resultados: hubo diferencias en la empatía en el factor curso. La empatía no se diferenció en el género. Conclusiones: los niveles de empatía, pasión son diferentes entre los cursos y no existen diferencias de empatía entre los géneros.

ABSTRACT Introduction: empathy is an important concept in the interaction between the Kinesiologist and the patient. Objective: to determine the levels of empathy in students of Kinesiology considering the variables course and gender from the School of Health Sciences, University of Atacama (Chile) in November 2018. Methods: exploratory and cross-sectional study. A sample of 191 students was included in the study. The Jefferson's Empathy scale was used. The median, quartile 1 and 3, inter-quartile difference and confidence interval of the median were estimated. The possible differences regarding empathy were studied using nonparametric tests (Mood Median). Results: there were differences in empathy in the course variable. Empathy was not different concerning gender variable. Conclusions: levels of empathy, along with the levels of interest showed differences between courses and there are no differentiation concerning empathy between genders.

Nucleolar Expression and Chromosomal Associations in Robertsonian Spermatocytes of Mus musculus domesticus.

We studied and compared the nucleolar expression or nucleoli from specific bivalents in spermatocytes of the standard Mus musculus domesticus 2n=40, of Robertsonian (Rb) homozygotes 2n = 24 and heterozygotes 2n = 32. We analyzed 200 nuclear microspreads of each specific nucleolar chromosome and spermatocyte karyotype, using FISH to identify specific nucleolar bivalents, immunofluorescence for both fibrillarin of the nucleolus and the synaptonemal complex of the bivalents, and DAPI for heterochromatin. There was nucleolar expression in all the chromosomal conditions studied. By specific nucleolar bivalent, the quantitative relative nucleolar expression was higher in the bivalent 12 than in its derivatives, lower in the bivalent 15 than in its derivatives and higher in the bivalent 16 than its Rb derivatives. In the interactions between non-homologous chromosomal domains, the nucleolar bivalents were preferentially associated through pericentromeric heterochromatin with other bivalents of similar morphology and sometimes with other nucleolar bivalents. We suggest that the nucleolar expression in Rb nucleolar chromosomes is modified as a consequence of different localization of ribosomal genes (NOR) in the Rb chromosomes, its proximity to heterochromatin and its associations with chromosomes of the same morphology.

Burkholderia pseudomallei ΔtonB Δhcp1 Live Attenuated Vaccine Strain Elicits Full Protective Immunity against Aerosolized Melioidosis Infection.

Burkholderia pseudomallei is a Gram-negative facultative intracellular bacterium and the causative agent of melioidosis, a severe infectious disease found throughout the tropics. This organism is closely related to Burkholderia mallei, the etiological agent of glanders disease which primarily affects equines. These two pathogenic bacteria are classified as Tier 1 select agents due to their amenability to aerosolization, limited treatment options, and lack of an effective vaccine. We have previously successfully demonstrated the immunogenicity and protective efficacy of a live attenuated vaccine strain, B. malleiΔtonB Δhcp1 (CLH001). Thus, we applied this successful approach to the development of a similar vaccine against melioidosis by constructing the B. pseudomalleiΔtonB Δhcp1 (PBK001) strain. C57BL/6 mice were vaccinated intranasally with the live attenuated PBK001 strain and then challenged with wild-type B. pseudomallei K96243 by the aerosol route. Immunization with strain PBK001 resulted in full protection (100% survival) against acute aerosolized melioidosis with very low bacterial burden as observed in the lungs, livers, and spleens of immunized mice. PBK001 vaccination induced strong production of B. pseudomallei-specific serum IgG antibodies and both Th1 and Th17 CD4+ T cell responses. Further, humoral immunity appeared to be essential for vaccine-induced protection, whereas CD4+ and CD8+ T cells played a less direct immune role. Overall, PBK001 was shown to be an effective attenuated vaccine strain that activates a robust immune response and offers full protection against aerosol infection with B. pseudomalleiIMPORTANCE In recent years, an increasing number of melioidosis cases have been reported in several regions where melioidosis is endemic and in areas where melioidosis had not commonly been diagnosed. Currently, the estimated burden of disease is around 165,000 new cases annually, including 89,000 cases that have fatal outcomes. This life-threatening infectious disease is caused by B. pseudomallei, which is classified as a Tier 1 select agent. Due to the high case fatality rate, intrinsic resistance to multiple antibiotic treatments, susceptibility to infection via the aerosol route, and potential use as a bioweapon, we have developed an effective live attenuated PBK001 vaccine capable of protecting against aerosolized melioidosis.

Evaluating New Compounds to Treat Burkholderia pseudomallei Infections.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease that requires long-term treatment regimens with no assurance of bacterial clearance. Clinical isolates are intrinsically resistant to most antibiotics and in recent years, isolates have been collected that display resistance to frontline drugs. With the expanding global burden of B. pseudomallei, there is a need to identify new compounds or improve current treatments to reduce risk of relapse. Using the Pathogen Box generated by Medicines for Malaria Venture, we screened a library of 400 compounds for bacteriostatic or bactericidal activity against B. pseudomallei K96243 and identified seven compounds that exhibited inhibitory effects. New compounds found to have function against B. pseudomallei were auranofin, rifampicin, miltefosine, MMV688179, and MMV688271. An additional two compounds currently used to treat melioidosis, doxycycline and levofloxacin, were also identified in the screen. We determined that the minimal inhibitory concentrations (MIC) for levofloxacin, doxycycline, and MMV688271 were below 12 µg/ml for 5 strains of B. pseudomallei. To assess persister frequency, bacteria were exposed to 100x MIC of each compound. Auranofin, MMV688179, and MMV688271 reduced the bacterial population to an average of 4.53 × 10-6% compared to ceftazidime, which corresponds to 25.1% survival. Overall, our data demonstrates that auranofin, MMV688197, and MMV688271 have the potential to become repurposed drugs for treating melioidosis infections and the first evidence that alternative therapeutics can reduce B. pseudomallei persistence.

Use of Reverse Vaccinology in the Design and Construction of Nanoglycoconjugate Vaccines against Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative, facultative intracellular pathogen that causes the disease melioidosis in humans and other mammals. Respiratory infection with B. pseudomallei leads to a fulminant and often fatal disease. It has previously been shown that glycoconjugate vaccines can provide significant protection against lethal challenge; however, the limited number of known Burkholderia antigens has slowed progress toward vaccine development. The objective of this study was to identify novel antigens and evaluate their protective capacity when incorporated into a nanoglycoconjugate vaccine platform. First, an in silico approach to identify antigens with strong predicted immunogenicity was developed. Protein candidates were screened and ranked according to predicted subcellular localization, transmembrane domains, adhesive properties, and ability to interact with major histocompatibility complex (MHC) class I and class II. From these in silico predictions, we identified seven "high priority" proteins that demonstrated seroreactivity with anti-B. pseudomallei murine sera and convalescent human melioidosis sera, providing validation of our methods. Two novel proteins, together with Hcp1, were linked to lipopolysaccharide (LPS) and incorporated with the surface of a gold nanoparticle (AuNP). Animals receiving AuNP glycoconjugate vaccines generated high protein- and polysaccharide-specific antibody titers. Importantly, immunized animals receiving the AuNP-FlgL-LPS alone or as a combination demonstrated up to 100% survival and reduced lung colonization following a lethal challenge with B. pseudomallei Together, this study provides a rational approach to vaccine design that can be adapted for other complex pathogens and provides a rationale for further preclinical testing of AuNP glycoconjugate in animal models of infection.

Loperamide misuse and abuse.

OBJECTIVE: The epidemic of opioid prescription deaths in recent years resulted in the recent rescheduling of hydrocodone-containing products to restrict access to them. Opioid users have recognized that loperamide can ameliorate withdrawal symptoms and also produce euphoria in very high doses. This article discusses the potential for loperamide misuse and abuse and examines trends in the increasing number of published cases of loperamide toxicity. DESIGN: PubMed was used to search MEDLINE for case reports of loperamide abuse. SETTING: United States. MAIN OUTCOME MEASURES: Numbers of cases of loperamide misuse, characteristics of patients, reported toxicities. RESULTS: From 1985 to 2016, 54 case reports of loperamide toxicity were published, with 21 cases between 1985 and 2013 and 33 cases between 2014 and 2016. In addition, 179 cases of intentional loperamide misuse were reported to the National Poison Database System between 2008 and 2016, with more than half reported after January 1, 2014. CONCLUSION: Loperamide misuse and abuse is increasing in the United States, and pharmacists are encouraged to monitor and restrict their sales.