Burkholderia pseudomallei, the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging, and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell surface polysaccharides and cell-associated and cell-secreted proteins. Based on those findings, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated, and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate, while immunization with Hcp1 and TssM produced high-titer IgG and robust gamma interferon-secreting T cell responses against the proteins. Extending upon these studies, we found that when mice were vaccinated with a combination of CPS-CRM197 and Hcp1, 100% of the mice survived a lethal inhalational challenge with B. pseudomallei. Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers, or spleens, indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights toward the development of a safe, affordable, and effective melioidosis vaccine.

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

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative organisms, which are etiological agents of glanders and melioidosis, respectively. Although only B. pseudomallei is responsible for a significant number of human cases, both organisms are classified as Tier 1 Select Agents and their diseases lack effective diagnosis and treatment. Despite a recent resurgence in research pertaining to these organisms, there are still a number of knowledge gaps. This article summarizes the latest research progress in the fields of B. mallei and B. pseudomallei pathogenesis, vaccines, and diagnostics.

Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ⌬tonB ⌬hcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain. Burkholderia mallei, the causative agent of glanders, is a Gramnegative, obligate mammalian pathogen. Glanders is primarily a disease of solipeds, with rare cases occurring among humans (1, 2). Naturally acquired human cases occur in areas of endemicity, particularly among those exposed to infected solipeds (2-4). Additionally, cases have been reported among laboratory workers (5, 6). The World Organization for Animal Health coordinates ongoing efforts toward worldwide eradication; however, regional endemicity still exists in Africa, Asia, the Middle East, and South America (2). Recent equid outbreaks in the Middle East and Asia (7-9) set the stage for possible glanders reintroduction into disease-free regions; as a result, glanders has been classified as a reemerging disease.Glanders is a debilitating and often fatal disease transmitted via cutaneous and respiratory routes. Disease course and severity are route dependent, with respiratory cases being the most severe (3). Respiratory infection is characterized by rapid onset of symptoms, including fever, lymphadenopathy, pulmonary abscesses, pneumonia, disseminated organ infection, and, ultimately, septicemia (3, 5, 10). Because of the high incidence of septicemia following respiratory infection, fatality rates in human respiratory cases have been estimated at 90% without treatment and 40% with aggressive antibiotic therapy (11).The use of B. mallei as a biothreat agent has been documented in different world military conflicts (10,12,13). Its amenability to aerosolization, low infectious dose, high case fatality rate, and high-level antibiotic resistance make B. mallei a top candidate for malevolent use (1, 10). Because of its perceived public health threat, the Department of Health and Human Services has categorized B. mallei as a tier 1 select agent. The lack of effective treatments against these bacteria highlights the need for an effective vaccine. Numerous vaccine strategies have been tested; however, to date there are n...

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.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a leading cause of foodborne illnesses worldwide and is a common serotype linked to hemorrhagic colitis and an important cause of hemolytic uremic syndrome (HUS). Treatment of EHEC O157:H7 infections is complicated, as antibiotics can exacerbate Shiga toxin (Stx) production and lead to more severe symptoms including HUS. To date, no vaccines have been approved for human use, exposing a void in both treatment and prevention of EHEC O157:H7 infections. Previously, our lab has shown success in identifying novel vaccine candidates via bio- and immunoinformatics approaches, which are capable of reducing bacterial colonization in an in vivo model of intestinal colonization. In this study, we further characterized 17 of the identified vaccine candidates at the bioinformatics level and evaluated the protective capacity of the top three candidates when administered as DNA vaccines in our murine model of EHEC O157:H7 colonization. Based on further immunoinformatic predictions, these vaccine candidates were expected to induce neutralizing antibodies in a Th2-skewed immunological response. Immunization of BALB/c mice with two of these candidates resulted in reduced bacterial colonization following EHEC O157:H7 challenge. Additionally, immune sera was shown to prevent bacterial adhesion in vitro to Caco-2 cells. Together, this study provides further validation of our immunoinformatic analyses and identifies promising vaccine candidates against EHEC O157:H7.

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