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 proteinand 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.KEYWORDS Burkholderia pseudomallei, melioidosis, nanoglycoconjugate, nanovaccine, reverse vaccinology B urkholderia pseudomallei is a Gram-negative aerobic bacterium common to tropical and subtropical climates worldwide. This saprophytic bacterium can survive in soil and water and, upon transmission to humans or other susceptible mammals, cause the disease melioidosis. Human disease can present with a wide variety of clinical manifestations, including cutaneous and soft tissue abscesses, lymphadenopathy, and sepsis (1, 2). Clinical symptoms have been correlated with the route of infection; percutaneous infection often results in a purulent lesion at the site of inoculation, whereas respiratory

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.

Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichia coli (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 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.

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.

The aim of this study was to identify and determine the prevalence of Eimeria species affecting sheep raised extensively in a semiarid region of Brazil. Fecal samples of native sheep were collected during the rainy and dry seasons. The degree of infection was determined by counting oocysts per gram (OPG) of feces, and the morphometric method was used for species identification. Oocysts were found in all the properties assessed, in which 68.3% of the animals were infected. The prevalence of oocysts was influenced by the season and animal category (P<0.05). It was higher during the rainy season than the dry season (80.2% vs. 55.8%) and highest in young animals than the adults animals (68.2% vs. 39.6%). The OPG was lower during the dry season (1,269 ± 312 vs. 4,400 ± 1,122). Ten species were found; of these, E. ovinoidalis, E. granulosa, E. faurei, and E. crandallis were the most frequent. E. ovinoidalis and E. crandallis were found in all properties, with their prevalences being 19.4% and 13.6% respectively. The high prevalence of pathogenic species shows that eimeriosis is a risk for animals raised extensively in the semiarid region.Keywords: Coccidiosis, morphometry, oocyts, OPG, parasitosis. ResumoObjetivou-se neste estudo identificar e determinar a prevalência de espécies de Eimeria que parasitam ovinos criados extensivamente em região semi-árida. Amostras de fezes de ovinos nativos foram coletados durante as estações chuvosa e seca. O grau de infecção foi determinado pela contagem de oocistos por grama de fezes (OoPG)e o método morfométrico foi utilizado para a identificação das espécies. Foram encontrados oocistos em todas os rebanhos avaliados e observou-se que 68,3% dos animais estavam infectados. A prevalência de oocistos foi influenciada pela estação climática e pela categoria dos animais (P<0,05), sendo mais alta durante a estação chuvosa em relação a estação seca (80,2% vs. 55,8%) e em animais jovens em relação aos animais adultos (68,2% vs. 39,6%). O OoPG foi menor durante a estação seca (1.269 ± 312 vs. 4.400 ± 1.122). Dez espécies foram encontradas sendo a E. ovinoidalis, E. granulosa, E. faurei, e E. crandallis as mais frequentes. E. ovinoidalis e E. crandallis foram encontrados em todas as propriedades, com prevalências de 19,4% e 13,6%, respectivamente. A alta prevalência de espécies patogênicas mostra que eimeriose é um risco para os animais criados extensivamente na região semiárida.

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.

Burkholderia pseudomallei (Bpm) is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, Bpm poses a significant health risk, especially in endemic regions, 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 Bpm 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 Bpm 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 Bpm to IECs, although the impact on full virulence was limited. Finally, we also showed that Bpm requires a functional T6SS for full virulence, bacterial dissemination, and for lethality in mice infected by the intragastric route. Overall, we showed that Bpm is an enteric pathogen, that type 1 fimbria is important for Bpm intestinal adherence and 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 Bpm.