Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

vaccines

Article Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

Michael H. Norris 1,2 ID , Mohammad S. R. Khan 1,2, Sunisa Chirakul 1,2, Herbert P. Schweizer 2,3 and Apichai Tuanyok 1,2,* 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine; University of Florida, Gainesville, FL 32608, USA; mhnorris@ufl.edu; (M.H.N.); siddiqur.r.khan@ufl.edu (M.S.R.K.); schirakul@ufl.edu (S.C.) 2 Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610, USA; hschweizer@ufl.edu 3 Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32603, USA * Correspondence: tuanyok@ufl.edu; Tel.: +1-352-294-8269

Received: 26 November 2017; Accepted: 6 January 2018; Published: 10 January 2018

Abstract: Burkholderia mallei is a host-adapted Gram-negative mammalian pathogen that causes the severe disease glanders. Glanders can manifest as a rapid acute progression or a chronic debilitating syndrome primarily affecting solipeds and humans in close association with infected animals. In USA, B. mallei is classified as one of the most important bacterial biothreat agents. Presently, there is no licensed glanders vaccine available for humans or animals. In this work, outer membrane vesicles (OMVs) were isolated from three attenuated biosafe bacterial strains, Burkholderia pseudomallei Bp82, B. thailandensis E555, and B. thailandensis TxDOH and used to vaccinate mice. B. thailandensis OMVs induced significantly higher antibody responses that were investigated. B. mallei specific serum antibody responses were of higher magnitude in mice vaccinated with B. thailandensis OMVs compared to levels in mice vaccinated with B. pseudomallei OMVs. OMVs derived from biosafe strains protected mice from acute lethal glanders with vesicles from the two B. thailandensis strains affording significant protection (>90%) up to 35 days post-infection with some up to 60 days. Organ loads from 35-day survivors indicated bacteria colonization of the lungs, liver, and spleen while those from 60 days had high CFUs in the spleens. The highest antibody producing vaccine (B. thailandensis E555 OMVs) also protected C57BL/6 mice from acute inhalational glanders with evidence of full protection.

Keywords: glanders; outer membrane vesicles; biosafe vaccines

1. Introduction The disease glanders is primarily a zoonotic disease of solipeds that in ancient times was recognized by both Hippocrates and Aristotle [1]. It is caused by the organism Burkholderia mallei, which is a host-adapted Gram-negative pathogen [2]. Historically, human cases of glanders were most often associated with individuals that were in close contact with infected horses, mules, or donkeys and was contracted through spreading of infectious secretions [3,4]. More recently, beginning in the 20th century and its two World Wars, B. mallei has been associated with biological warfare, intentional misuse, and laboratory acquired infections [5–7]. It was used as a biological weapon in World War I by Germany and the Japanese in World War II researched human infections and intentional contamination of public water sources. Following WWII, the USSR conducted ﬁeld tests and anecdotal evidence suggests usage in Afghanistan. The disease has been eradicated in North America, Australia, and most of Europe through careful surveillance, culling of infected animals and importation practices [8,9]. The modern era has ushered in the global movement of livestock leading to increased cases in other parts of the world and the labeling of glanders as a re-emerging threat [10].

Vaccines 2018, 6, 5; doi:10.3390/vaccines6010005 www.mdpi.com/journal/vaccines Vaccines 2018, 6, 5 2 of 20

Generally, the disease itself can manifest as an acute or chronic infection. The acute disease presents as a localized cutaneous infection (also known as farcy) or pulmonary infection that can progress to either septicemia or severe pneumonic disease thereafter [4].Estimated incubation periods for acute infections may vary from 1–14 days and if left untreated can lead to death within the same period [11]. Chronic infections are indicated by chronic nasal discharge, chronically enlarged lymph nodes or lymphatic vessels, and in the case of farcy, numerous cutaneous ulcerations [2,3]. Chronic disease onset can occur up to 12 weeks after exposure, and if untreated, leads to death [4,11]. The organism is highly infectious with reports in the animal model estimating the LD50 at between 2 and 820 CFU depending on the strain of B. mallei, route of infection and rodent model utilized [9,12–14]. The United States Centers for Disease Control and Prevention (CDC) considers the organism as a potential threat to human health while the United States Department of Agriculture (USDA) considers it a threat to animal health and so B. mallei is listed as a Tier 1 overlap select agent. A vaccine against glanders is currently unavailable and treatment remains difficult. B. mallei shares close genetic homology to the fellow select agent B. pseudomallei, the etiological agent of melioidosis, and has been determined to be a species borne of B. pseudomallei by host-adaptation and gene loss [15,16]. Burkholderia thailandensis is similar to B. pseudomallei and B. mallei but is relatively avirulent with LD50 levels of ~106 CFU in the susceptible Syrian hamster model [17,18]. High genetic homology and similar mechanisms of molecular pathogenesis within the three species mean they share numerous antigenic determinants. Important virulence factors such as the major type 3 secretion system (T3SS) Burkholderia secretion apparatus (Bsa) [19], type 6 secretion system 1 (T6SS-1) [20–22], Burkholderia intracellular motility protein A (BimA) [23] and lipopolysaccharide (LPS) are conserved [24,25]. B. thailandensis lacks the T3SS-3 that is present in B. mallei and B. pseudomallei. There are differences in O-antigen modifications of B. mallei LPS [26,27] and most B. thailandensis strains lack the major capsular polysaccharide possessed by B. mallei and B. pseudomallei [28]. Exceptions to the rule have been discovered, where environmental isolates of B. thailandensis strains with a capsule have been identified but remain highly attenuated [29]. A recent paper has demonstrated that CPS isolated from one such strain, (B. thailandensis strain E555) can afford significant protection to mice challenged intraperitoneally with B. pseudomallei K96243 [30]. For the most part, close genetic and antigenic homology means that material from any of the three species can be used to vaccinate against glanders or melioidosis but does not discount the possibility that species-specific antigens might be absent [31–33]. An effective vaccine would induce high levels of protection and be safe and easy to produce. Live-attenuated vaccines for glanders have been produced and can protect mice from acute glanders [14,34]. The major drawback for live vaccines is that safety and efficacy testing in humans is prohibitive. The numerous antigens present in whole cells are an advantage that subunit vaccines lack. Heat-killed vaccines also contain numerous antigens but are considered too risky for human vaccination. Effective glycoconjugate and nanoparticle-based vaccines have also been produced but may be limited by the minimal amount of antigens that are present in the formulations [35–38]. An alternative is the production of outer membrane vesicles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria under stress conditions such as antibiotic treatment or nutrient deprivation during stationary growth [39]. The outer membrane blebs off as vesicles that contain many proteins and polysaccharides associated with the outer membrane of the bacterium (and less frequently periplasmic, inner membrane and cytoplasmic in decreasing order) but are non-viable [40,41]. OMV producing bacteria can be engineered to optimize protein cargo present, thus allowing a fine-tuning of the immune responses generated. The EMA and FDA approved the vaccine Bexsero, which includes OMVs as adjuvant, for prevention of meningitis in humans [42]. OMVs have previously been used to protect mice against lethal melioidosis [43,44]. In the previously published works OMVs were isolated from virulent B. pseudomallei. The use of an OMV based vaccine has not been evaluated for glanders but the requirement for both significant humoral and cell-mediated immunity in protection from glanders makes OMVs an attractive vaccine candidate. OMVs also fulfill Vaccines 2018, 6, 5 3 of 20 the safety aspects associated with future applications involving humans. To meet and exceed these requirements, OMVs can be isolated from biosafe surrogate species. In this work, OMVs were isolated from three biosafe source strains and characterized for presence of polysaccharide antigens. The OMVs were used to vaccinate BALB/c mice and levels of OMV-specific antibodies were investigated. The magnitude and nature of antibody responses to B. mallei and B. pseudomallei cell lysates and opsonophagocytic ability were measured. BALB/c mice were challenged with virulent type strain B. mallei China 7 by the intranasal route and vaccine efficacy evaluated as indicated by prolonged survival. Organ bacterial burden in survivors was also determined. C57BL/6 mice were vaccinated with the best candidate identified in BALB/c mice and protection in combination with sterilizing immunity was assessed. This work represents a step forward in the development of a safe and effective vaccine against acute lethal glanders with the indication of chronic glanders prevention in the form of sterilizing immunity.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions All Select Agent work was carried out in CDC/USDA Tier 1 select agent approved BSL-3 and ABSL-3 facilities at the University of Florida following Tier 1 regulations. All protocols were approved by the Institutional Biosafety Committee prior to implementation under numbers BA-4200 and RD-4158. B. mallei strains (China 7, G-2(3), India 86-567-2, Ivan, SAVP1, and Turkey #1; (obtained from the CDC)), B. pseudomallei 1026b (CDC/USDA registered in house bacterial inventory) and B. thailandensis strains (E555, obtained from Mahidol University; and TxDOH, obtained from CDC) were grown on LB Lennox broth or agar (5 g/L NaCl) (LB, Fisher BioReagents, Pittsburgh, PA, USA) and grown at 37 ◦C. LB broth was used for liquid growth of all strains. B. mallei strains were grown on or in LB media with the addition of 4% (v/v) glycerol (LB4G). Select Agent excluded strain Bp82 [45] and was grown on LB or TSA with 0.6 mM adenine (Amresco, Solon, OH, USA). The mouse cell line RAW264.7 (American Type Culture Collection, ATCC) was grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM)-high ◦ glucose + L-glutamine (HyClone) with 10% FBS (HyClone) in 5% CO2 at 37 C. All plastic ware was Corningware with CellBIND surface. Culturing cells was carried out essentially as described previously [46–51].

2.2. Stains, Western Blots and ELISA Whole cell lysates were produced by heating equivalent amounts of live bacteria in 1× PBS at 110 ◦C for 15 min in a digital heat bath. Select-agent bacteria were prepared in the same manner by a validated and verified protocol to ensure sterility. Sample preparation was carried out as previously described with or without Proteinase K digestion depending on the gel [27]. Samples were mixed with 2× LPS lysis/loading buffer prior to running on a gel. SDS-PAGE was carried out using homemade polyacrylamide gels with a 12% resolving gel and a 4% stacking gel. Coomassie blue stains were carried out using standard procedures. Silver stains of SDS-PAGE gels were done according to the manufacturer’s instructions using the Pierce™ Silver Stain Kit (ThermoFisher, Waltham, MA, USA). Colorimetric Western blots were performed by semi-dry electroblotting of SDS-PAGE run gels onto methanol soaked Immobilon PSQ PVDF membranes from Millipore™ (Burlington, MA, USA) or Odyssey nitrocellulose membranes soaked in buffer from LI-COR™ (Lincoln, NE, USA). Blots were washed with 1× PBS, blocked with 1% skim milk in PBS and detected following manufacturer’s instructions with 1-Step™ Ultra TMB-Blotting Solution (ThermoFisher, Waltham, MA, USA). ELISAs were carried out as previously described [50]. Briefly, Immulon 4HBX flat bottom plates were coated with 100 µL antigen at 100 µg/mL suspended in PBS overnight at room temperature. This concentration of antigen was determined to give the best dynamic range in checkerboard optimization ELISAs. The plates were washed three times with PBS/T buffer, blocked with 5% skim milk in PBS/T for 1 h and washed three times with PBS/T buffer again. Detection was carried out by incubating for 1 h with Vaccines 2018, 6, 5 4 of 20 peroxidase conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:1000 in blocking solution. After 1 h, wells were washed 3 times with PBS/T then detected with 1-Step™ Ultra TMB-ELISA solution (ThermoFisher, Waltham, MA, USA). The reaction was stopped by adding 1 M H3PO4 and the absorbance at 450 nm was measured.

2.3. Purification of OMVs OMVs were purified according to Kulp and Kuehn [52] with some modifications. Starter cultures were grown in 10 mL LB or, if Bp82, LB + adenine overnight and used to inoculate 1 L of the same media as a 1:100 dilution. Cultures were grown at 37 ◦C in an orbital shaker using 4 L Erlenmeyer flasks to late stationary phase. Upon reaching an optical density at 600 nm (OD600 nm) of ~4.0 (at 48–72 h) cells were harvested by centrifugation at 8000× g for 20 min at 4 ◦C. The supernatant was passed consecutively through first a 0.45 µm filter unit and then a 0.22 µm unit (Millipore). The filtered supernatant was centrifuged at 38,000× g for 2 h at 4 ◦C. The pellet was resuspended in 1 mL of PBS and 10% was plated for sterility assurance. Samples were submitted to gradient centrifugation in OptiPrep 45–20% (Sigma) at 280,000× g for 3 h at 4 ◦C. Each layer was analyzed by SDS-PAGE and layers containing OMV material (25% and 30% OptiPrep layers) were combined and the samples were lyophilized and dry weights determined. OMV preparations were suspended to 10 mg/mL in PBS and stored at −80 ◦C for further analysis.

2.4. Transmission Electron Microscopy of OMVs OMVs were diluted and allowed to dry on a hydrophobic Formva ﬁlm—coated copper grid and negatively stained with 2% uranyl acetate. For CPS labeling, the primary antibody was the mouse anti-CPS IgG mAb 4C4 (kindly provided by Dr. David AuCoin at University of Nevada, Reno). The secondary antibody was goat anti-mouse IgG (Electron Microscopy Sciences, Hatﬁeld, PA, USA) conjugated to a 12 nm gold particle and negatively stained with 1% uranyl acetate. Images were captured at 100 kV with Hitachi H-7000 transmission electron microscope (Hitachi, Chiyoda, Tokyo, Japan) at the UF Interdisciplinary Center for Biotechnology Research Electron Microscopy Core.

2.5. MALDI-TOF Scans of Purified Lipid A LPS was isolated from strains using a modified hot-phenol extraction done essentially as described [50,51,53]. Strains were grown on 8–10 plates of TSA or LB-agar (with adenine in the case of Bp82) for 72 h. Lawns were resuspended in PBS and collected in 2 mL gasketed microcentrifuge tubes and heat-killed at 110 ◦C for 15 min. Phenol was added to the lysed solution to a final concentration of 50%. Samples were dialyzed until free of phenol using tubing with 12–14 kDa molecular weight cutoff against distilled water for 3–5 days. The samples were treated with DNase I for 2 h, RNase h for 2 h, and Proteinase K overnight, then further purified as previously described [53]. Samples were weighed and resuspended in 2 mL of endotoxin free water. For isolation of lipid A, 2 mg of each LPS samples were treated with 1 M acetic acid for 1.5 h on a 100 ◦C heat-block essentially as previously described [54]. Samples were lyophilized and resuspended in 100 µL of water using a sonicating water bath for 10 min. Lipid As were purified by precipitation with 500 µL of acidified ethanol (1% HCl in 100% ethanol), centrifuged at 12,000× g and 4 ◦C for 10 min, twice washed with non-acidified 100% ethanol, and then dried under vacuum. For MALDI-TOF/TOF analysis, dried samples were prepared as previously described [51] in a matrix of 500 mM 2,5-dihydribenzoic acid [54,55]. Mass spectra were acquired with a 4700 MALDI-TOF/TOF analyzer (ABSciex, Framingham, MA, USA) and a Nd:YAG (neodymium-doped yttrium aluminum garnet) laser with a 200-Hz sampling rate. The instrument was operated in the negative ion reflector mode, using a fixed laser intensity of 2500 to accumulate 1000 shots/spectrum across the mass range of 1200–1900 Da. Acquired spectra T2D files were converted to mzXML files and further analyzed on the open source mMass software. Three independently isolated lipid A samples had their mass scans normalized and averaged for the spectrums presented. Vaccines 2018, 6, 5 5 of 20

2.6. Animal Experiments and Ethical Statement Select Agent animal work was performed in a CDC/USDA Tier 1 approved facility at the University of Florida following Tier 1 regulations. Animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida before work began. Female BALB/c mice and C57BL/6 mice between 4 and 6 weeks of age were purchased from Charles River Laboratories (Wilmington, MA, USA). OMV material was diluted to 25 µg/mL in PBS. The prime vaccine doses of 100 µL containing 2.5 µg OMV were inoculated subcutaneously by syringe. Two weeks later the animals were boosted with the same dose and the second boost was given two weeks after that. Two weeks after the last boost mice were tail vein bled for serum and four weeks after the last boost, mice were moved into micro-isolator cages under pathogen-free conditions for challenge at BSL3. The B. mallei China 7 inoculate was grown overnight in LB4G media and frozen in 20% glycerol aliquots at −80 ◦C. Forty-eight hours later, an aliquot was thawed, diluted, and plated for CFU enumeration on LB4G agar medium. Dilution values were determined for the target inoculation of 1.5 × 104 CFU (22 × LD50 for BALB/c mice [33] and 17 × LD50 for C57BL/6 mice [56]) in 20 µL. Animals were anesthetized with 87.5 mg/kg of ketamine (Patterson Veterinary) of body weight plus 12.5 mg/kg xylazine. Once fully anesthetized, groups of 5 mice (n = 5) were challenged with the 20 µL inoculum by pipetting into the nares of the mouse alternating nostrils until fully inhaled. Mice were observed twice daily for the ﬁrst 4 days then once daily until the end of the studies. Mice were euthanized when moribund or at the end of the study. Organs were processed as follows. The lungs, livers, and spleens from survivors were isolated and organs were homogenized in 5 mL of PBS using a stomacher (Seward, AK, USA). Undiluted and diluted aliquots were plated on LB4G for CFU determination after 48 h growth at 37 ◦C. Colonies were positively identiﬁed as B. mallei by testing with the latex agglutination test as previously described [57,58].

2.7. Characterization of Antibody Responses ELISAs for detection of anti-OMV antibody induction in OMV-vaccinated and vehicle only mice were carried out by coating each well overnight with 100 µg/mL of OMV in PBS. This amount of OMV was determined to give us the best dynamic range from optimization ELISAs so that even low level antibody responses could be detected. Serum samples were serially diluted starting at 1:20 for OMV ELISAs in blocking solution and incubated in the plates for 1 h following the protocol above. For ELISAs measuring relative OMV induced anti-B. mallei or B. pseudomallei antibody responses, 50 ng equivalents (optimally determined by checkerboard assay) of heat-killed B. mallei China 7 or B. pseudomallei 1026b of known CFU were used to coat plates assuming a single bacterium has a mass of 10−12 g. Total bound mouse serum at 1:500 dilution (optimally determined by checkerboard assay) was detected with goat anti-Mouse IgG/IgA/IgM (H + L)-HRP (Thermo Fisher) and developed as described above. Numbers presented are in Absorbance units to allow direct correlation of total antibody production by combined measure of all isotypes. Characterization of B. mallei IgG responses induced by OMV vaccination or vehicle (PBS) only was done using B. mallei China 7 lysates as described above but represented as reciprocal endpoint titers because single isotypes were being measured. Serum dilution was started at 1:200 in blocking solution and serially diluted to 1:25,600. Bound IgG1 was detected with goat anti-mouse IgG1-HRP secondary (Thermo Fisher) and bound IgG2a was detected with goat anti-mouse IgG2a-HRP (Thermo Fisher); both at 1:1000 dilution in blocking solution. The endpoint titer was determined as the dilution above the cutoff where the cutoff is three standard deviations above the mean of all PBS serum measurements. If there was no observable reaction at 1:200, a dilution factor of 1:100 was arbitrarily assigned as the cutoff. Results are expressed as the individual reciprocal endpoint titers or as the ratio of the geometric mean reciprocal endpoint IgG1 and IgG2a titers. Vaccines 2018, 6, 5 6 of 20

2.8. Opsonophagocytosis Assay The opsonophagocytosis assay was performed by diluting B. mallei China 7 strain grown in ◦ 8 LB4G medium overnight at 37 C in PBS to an OD600 nm of 1, empirically determined to be ~5 × 10 B. mallei CFU. The cultures were then diluted down to 5 × 106 CFU/mL in DMEM or DMEM + 0.2% heat-inactivated (56 ◦C for 30 min) pooled mouse serum in Hyclone™ Dulbecco’s Modiﬁed Eagle Medium (DMEM) (GE Healthcare Life Sciences, Chicago, IL, USA). The dilutions were used to infect RAW264.7 macrophages in 24-well CellBIND plates (Corning, Corning, NY, USA) at an MOI of 10:1. After 1 h, the bacteria-containing medium was removed and the monolayers were washed three times with pre-warmed PBS and fresh DMEM containing 250 µg/mL of kanamycin was added to suppress extracellular bacterial growth. At 2 h post infection the media was removed, monolayers were washed 2 times with PBS and lysed by adding 1 mL of 0.2% Triton-X100 in PBS for 15 min. The lysate was diluted, plated onto LB4G plates and incubated at 37 ◦C for 48 h to enumerate colonies. Intracellular CFU were calculated and invasion efﬁciency was determined by dividing the intracellular CFU by the initial number of infecting bacteria. The experiment was carried out in quadruplicate and the numbers represent the average of all four replicates with the error bars representing the SEM. Data shown is representative of two independent experiments.

2.9. Statistical Analysis Signiﬁcant differences in antibody responses to whole cell lysate and intracellular CFU in the opsonophagocytosis assay between all groups was determined by ordinary one-way ANOVA using Tukey’s multiple comparisons test where ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Statistically signiﬁcant increases in survival were determined by Log-rank (Mantel-Cox) test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

3. Results

3.1. Purification and Characterization of OMVs OMVs were purified from two B. thailandensis strains, E555 and TxDOH. Both strains are known to produce a similar B. pseudomallei or B. mallei capsular polysaccharide. Strain E555 produces the same O-antigen structures as those found in B. pseudomallei. TxDOH harbors a point mutation in oacA resulting in a truncated OacA protein. Because OacA is required for 4-O acetylation its LPS O-antigen most likely lacks the 4-O acetylation of the talose residue similar to the B. mallei LPS O-antigen [27]. A biosafe B. pseudomallei strain Bp82, an attenuated select agent excluded derivative of B. pseudomallei 1026b [45], was also used to isolate OMVs. The previously published methods used to isolate OMVs from bacteria in exponential phase for melioidosis vaccination [43,44] were not ideal in our hands but served as an excellent starting point. In this work a less delicate method involving growth of bacteria to stationary phase, pelleting of the bacteria, two-step filtration, and density gradient centrifugation produced consistent results. Transmission electron microscopy (TEM) images of the crude OMV preparations show numerous OMVs and flagellar fragments present (Figure1A,B). Figure1C–E shows that polysaccharides present in the cell lysates are also present in the OMV preparations. Silver stains, LPS, and CPS Western blots show that the biosafe strain-derived OMV preparations contain the major capsular polysaccharide, CPS, and LPS. Of particular note is the strong signal from the TxDOH O-antigen in Figure1E, lanes TxDOH, CL and OMV. The 4C7 mAb has a high affinity to the talose residue of the O-antigen without the acetylation [59]. 4 O-acetylation of this site, as in Bp82 and E555 LPS, results in reduced binding affinity by mAb 4C7. Serum from a rhesus macaque 28 days post-challenge with aerosolized B. pseudomallei strain 1026b shows intense reactivity to mainly the LPS O-antigen in all cell lysates from the OMV parent strains and OMV preparations (Figure1F). This particular macaque demonstrated very low background levels of anti-LPS specific antibodies prior to aerosol challenge that increased post-exposure, indicative of a highly specific Western blot (AppendixA Figure A1). Coomassie blue Vaccines 2018, 6, 5 7 of 20 Vaccines 2018, 6, 5 7 of 20 staininglysates of indicate the equivalent higher molecular amounts weight of OMVs proteins and cell predominate lysates indicate the Bp82 higher and molecular E555 OMV weight preparation proteins predominatewhile a wider the range Bp82 of and proteins E555 OMV are present preparation in the whileTxDOH a widerOMV preparations range of proteins (Appendix are present A Figure in the TxDOHA2). OMV preparations (AppendixA Figure A2).

A Crude OMVs B

C D E F Bp82 E555 TxDOH Bp82 E555 TxDOH Bp82 E555 TxDOH Bp82 E555 TxDOH CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV CL OMV kDa 245

100

11 Silver CPS mAb 4C4 LPS mAb 4C7 NHP serum

FigureFigure 1. Analysis1. Analysis of outerof outer membrane membrane vesicles vesicles (OMVs) (OMVs) and and immunogenic immunogenic polysaccharides. polysaccharides. (A) Crude(A) OMVCrude negative OMV stainednegative TEM stained image TEM showing image numerousshowing numerous vesicles and vesicles flagellar and material. flagellar (Bmaterial.) Zoom image(B) ofZoom crude image OMV of from crude the OMV area indicatedfrom the ar byea the indicated white boxby the in (whiteA). (C box) Silver in (A stain). (C of) Silver polysaccharide stain of preparationspolysaccharide from preparations cell lysates from (CL) cell and lysates OMVs. (CL) and (D) OMVs. Western (D blot) Western of the blot major of theB. major mallei B.capsular mallei capsular polysaccharide (CPS) reveals the high molecular weight polysaccharide is present in all polysaccharide (CPS) reveals the high molecular weight polysaccharide is present in all three biosafe three biosafe strains and their OMVs. (E) Western blot of the LPS O-antigen from all three biosafe strains and their OMVs. (E) Western blot of the LPS O-antigen from all three biosafe strains and their strains and their OMVs using B. mallei O-antigen specific mAb 4C7. (F) Reactivity of non-human OMVs using B. mallei O-antigen specific mAb 4C7. (F) Reactivity of non-human primate (NHP) serum primate (NHP) serum with cell lysate and OMVs from all three biosafe strains. with cell lysate and OMVs from all three biosafe strains. Further refinement of the association between CPS and the B. thailandensis OMV membranes wasFurther accomplished refinement by TEM of theimmunogold association labeling between of CPS CPS using and the the CPS-specificB. thailandensis mAbOMV 4C4 as membranes primary wasantibody accomplished [60]. The by black TEM dots immunogold surrounding labeling the OMVs of CPS indicate using large the CPS-specificamounts of CPS mAb associated 4C4 as primary with antibodyOMVs from [60]. Bp82 The blackand the dots B. surroundingthailandensis OMVs the OMVs (Figure indicate 2A–C insets large amountsand chevrons). of CPS Visible associated in these with OMVsTEM fromimages Bp82 of the and crude the B. preparations thailandensis areOMVs flagella (Figurer fragments2A–C insetsand OMVs and chevrons).of different Visible diameters. in these It TEMhas imagesbeen shown of the that crude most preparations B. pseudomallei are LPS flagellar are weaker fragments activators and of OMVs innate ofimmunity different [51,61,62] diameters. It hascompared been shown to those that from most B. thailandensisB. pseudomallei. LPSLPS was are isolated weaker from activators the three of biosafe innate strains immunity used [ 51in, 61the,62 ] comparedstudy and to thosethe molecular from B. thailandensis masses of . LPSthe hydrol was isolatedyzed lipid from A the fragments three biosafe were strains measured used inby the studyMALDI-TOF and the molecular and compared masses (Figure of the 2D–F). hydrolyzed The lipid lipid A Amoieties fragments from were E555 measuredand TxDOH by have MALDI-TOF more andpenta-acylated compared (Figure lipid A2D–F). than the The Bp82 lipid lipid A moieties A and do from not E555contain and the TxDOH additional have hydroxyl more penta-acylated groups that

Vaccines 2018, 6, 5 8 of 20

Vaccines 2018, 6, 5 8 of 20 lipid A than the Bp82 lipid A and do not contain the additional hydroxyl groups that are found in B.are pseudomallei found inlipid B. pseudomallei A, including lipid Bp82. A, Bothincluding characteristics Bp82. Both are characteristics associated with are increased associated ability with to induceincreased innate ability immunity to induce as innate compared immunity to less as penta-acyl compared lipidto less A penta-acyl and presence lipid ofA and hydroxyl presence groups, of ashydroxyl in the Bp82 groups, lipid A.as Followingin the Bp82 density lipid A. gradient Following purification density gradient TEM analysis purification of vaccine TEM analysis preparations of indicatedvaccine highlypreparations pure OMVindicated free ofhighly flagellar pure contamination.OMV free of flagellar Contribution contamination. of flagellar Contribution contamination of toflagellar protective contamination responses was to protective deemed negligibleresponses was as B. deemed mallei isnegligible non-motile as B. and mallei previously is non-motile published and datapreviously show that published including data the flagellarshow that component including FliCthe inflagellar a glanders component nanoparticle FliC in vaccine a glanders did not enhancenanoparticle post-challenge vaccine did survival not enha ornce antibody post-challenge responses survival compared or antibody to a non-specificresponses compared TetHc protein to a componentnon-specific [35 TetHc]. protein component [35].

A B C

Bp82 E555 TxDOH D E F 4-1P 4-1P E555 Bp82 4-1P Bp82 5-2P 5-2P 5-2P

5-1P 5-1P 5-1P -OH -OH -OH -OH -OH -OH

TxDOH E555 TxDOH

Figure 2. Transmission electron microscopy of capsular polysaccharides and lipid A moieties present Figure 2. Transmission electron microscopy of capsular polysaccharides and lipid A moieties present in OMV production strains. (A–C) TEM images of Bp82, E555, and TxDOH-derived OMVs in OMV production strains. (A–C) TEM images of Bp82, E555, and TxDOH-derived OMVs (chevrons), (chevrons), insets are zoomed images of CPS (mAb 4C4 specific immunogold labeling; black dots) in insets are zoomed images of CPS (mAb 4C4 speciﬁc immunogold labeling; black dots) in close close association with the OMVs. (D–F), MALDI-TOF/TOF mass scans of lipid A purified from the association with the OMVs. (D–F), MALDI-TOF/TOF mass scans of lipid A puriﬁed from the indicated indicated strains. Flagellar fragments are indicated by caret symbols. (D) E555 (top, olive) compared strains. Flagellar fragments are indicated by caret symbols. (D) E555 (top, olive) compared to TxDOH to TxDOH (bottom, gold) lipid A. (E) Bp82 (top, cyan) compared to E555 (bottom, olive) lipid A. (F) E F (bottom,Bp82 (top, gold) cyan) lipid compared A. ( ) to Bp82 TxDOH (top, (bottom, cyan) comparedgold) lipid toA. 4-1P, E555 mass (bottom, peak olive)of tetra-acylated lipid A. (singly) Bp82 (top,phosphorylated cyan) compared lipid toA; 5-1P, TxDOH mass (bottom, peak of penta-ac gold) lipidylated A. singly 4-1P, phosphorylat mass peak ofed tetra-acylatedlipid A; 5-2P, mass singly phosphorylatedpeak of penta-acylated lipid A; 5-1P,doubly mass phosphorylated peak of penta-acylated lipid A; -OH, singly indica phosphorylatedtes additional hydroxyl lipid A; 5-2P, groups mass peaknot of present penta-acylated in B. thailandensis doubly phosphorylatedlipid A moieties. lipid A; -OH, indicates additional hydroxyl groups not present in B. thailandensis lipid A moieties. 3.2. Strong Antibody Responses Are Generated by OMV Vaccination 3.2. Strong Antibody Responses Are Generated by OMV Vaccination Following the prime-boost-boost vaccination regimen with PBS and the different OMVs, mouse serumFollowing was collected the prime-boost-boost and ELISAs were vaccination run to compar regimene relative with antibody PBS and responses the different to the OMVs, vaccination mouse serummaterial was (Figure collected 3A,B). and PBS ELISAs control were serum run tohad compare very low relative reactivity antibody to each responses material while to the vaccinated vaccination materialmice had (Figure high3 A,B).levels PBS of controlserum antibody serum had to veryeach low respective reactivity OMV to each preparation. material whileThe reciprocal vaccinated miceendpoint had high titers levels that ofwere serum three antibody standard to deviat each respectiveions above OMVthe PBS preparation. vaccinated Themean reciprocal for Bp82, endpoint E555, titersand that TxDOH were threevaccinated standard mouse deviations serum were above 320, the PBS2560, vaccinated and 1280, meanrespectively. for Bp82, This E555, indicated and TxDOH an vaccinatedeight-fold mouse higher serumantibody were response 320, 2560, generated and 1280, by respectively.E555 OMVs and This four-fold indicated higher an eight-fold response higher by TxDOH OMVs compared to Bp82 OMVs. antibody response generated by E555 OMVs and four-fold higher response by TxDOH OMVs compared to Bp82 OMVs.

Vaccines 2018, 6, 5 9 of 20 Vaccines 2018, 6, 5 9 of 20

A Bp82 OMV coated E555 OMV coated TxDOH OMV coated 3

Vehicle control 2 Bp82 OMV E555 OMV TxDOH OMV 1

Absorbance units (Au) units Absorbance 0 l V V o rol ntr OMV OM o OM control 2 c cont H 8 555 O e le E e D icl Bp c icl h Tx eh ehi e V V V Vaccine B

Bp82 OMV 3 E555 OMV TxDOH OMV 2

Absorbance units (Au) 0 0 0 0 0 0 0 0 20 8 6 2 4 8 60 1: 1:40 1:8 :160 32 :640 2 5 1 2 4 9 1 1: 1 :1 :2 :5 0 0 0 1 1 1 :1 2 4 1 1: 1: Serum dilution

Figure 3.3.Stronger Stronger OMV OMV speciﬁc specific antibodies antibodies are produced are after produced vaccination after with vaccinationB. thailandensis with-derived B. thailandensisOMVs. (A) Average-derived reactivity OMVs. ( ofA) serially Average diluted reactivity serum of from serially OMV-vaccinated diluted serum mice from to OMV-vaccinated the immunogenic micematerial to the is speciﬁcimmunogenic compared material to serum is specific from vehiclecompared control-vaccinated to serum from mice.vehicle (B control-vaccinated) Endpoint serum mice.dilution (B) compared Endpoint between serum dilution OMV vaccinated compared groups between measured OMV vaccinated by ELISA and groups represented measured as absorbance by ELISA andunits represented at 450 nm. as absorbance units at 450 nm.

3.3. Characterization of B. mallei Sp Specificecific Antibody Responses to OMVs To compare the magnitude of B. mallei cross-reactive antibody production, serum samples were run againstagainst heat-killed heat-killed lysate lysate from fromB. mallei B. malleiChina China 7 and bound7 and mousebound immunoglobulinmouse immunoglobulin was measured. was measured.It was found It thatwas subcutaneousfound that subcutaneous vaccination vaccin using TxDOHation using OMVs TxDOH produced OMVs the produced strongest the anti- strongestB. mallei anti-antibodyB. mallei response, antibody significantly response, more significantly than E555 more OMVs than (Figure E5554A). OMVs Both B.(Figure thailandensis 4A). -derivedBoth B. thailandensisOMVs generated-derived significantly OMVs generated higher anti- significantlyB. mallei antibody higher responsesanti-B. mallei than antibody Bp82 OMVs, responses which werethan Bp82not significantly OMVs, which different were not than significantly PBS vaccinated different mouse than sera. PBS vaccinated Specificity mouse of the antibodysera. Specificity response of theto B. antibody mallei was response measured to B. mallei by running was measured the same by samples running against the sameB. samples pseudomallei against1026b B. pseudomallei heat-killed 1026blysate (Figureheat-killed4B). lysate In contrast (Figure to the4B). observation In contrast into Figurethe observation4A, anti- B. in pseudomallei Figure 4A, antibodyanti-B. pseudomallei responses antibodygenerated responses by TxDOH generated OMVs wereby TxDOH not significantly OMVs were different not significantly than those different in PBS than vaccinated those in mice. PBS vaccinatedE555 OMVs mice. generated E555 high OMVs levels generated of anti-B. high pseudomallei levels ofantibody anti-B. responsespseudomallei in additionantibody to responses anti-B. mallei in additionantibody to responses. anti-B. mallei antibody responses.

Vaccines 2018, 6, 5 10 of 20 Vaccines 2018, 6, 5 10 of 20

Serum Ab to A B. mallei China 7 lysate C B. mallei specific IgG1 *** **** **** 3 **** 5 **** 10 ** ns 104 2 103 1 ns 102

0 1

Absorbance units (Au) units Absorbance 10 l o V V V V V V rol M M M Reciprocal endpoint titer t M ntr O OM O OM o O on c 2 O 5 H c 2 5 H 8 5 p8 e O B E55 l Bp E5 D icle ic h TxDO Tx eh Ve V Vaccination Vaccination

Serum Ab to B B. pseudomallei 1026b lysate D B. mallei specific IgG2a

**** ** 3 5 **** 10 * 104 2 ns ns ns 103 1 ns 102

0 101 Absorbance units (Au) units Absorbance V V

MV titer endpoint Reciprocal trol n OM ontrol O o OMV 2 OMV 5 c 2 5 c 8 OH OM 8 5 le D e p c Bp E55 x l B E5 i T TxDOH OMV Veh Vehic Vaccination Vaccination

Figure 4. Biosafe strain-derived OMVs produce B. mallei and B. pseudomallei specific serum antibody Figureresponses. 4. Biosafe (A) Serum strain-derived antibody responses OMVs produce in vehicle B. mallei only or and OMV-vaccinated B. pseudomallei micespecific (PBS serum n = 5, antibody all others responses.n = 10) to B.(A mallei) SerumChina antibody 7 lysate. responses (B) Serum in antibodyvehicle only responses or OMV-vaccinated in vehicle only mice or OMV-vaccinated (PBS n = 5, all othersmice to nB. = pseudomallei 10) to B. 1026bmallei lysate China (PBS 7 lysate. n = 5, all (B others) Serum n = 10).antibody Measured responses by ELISA in and vehicle represented only or as OMV-vaccinatedabsorbance units atmice 450 to nm. B. ( Cpseudomallei,D) Serum IgG11026b and lysate IgG2a (PBS responses n = 5, inall vehicle others only n = or10). OMV-vaccinated Measured by ELISAmice (PBS and nrepresented = 5, all others as absorbance n = 10) to B. units mallei at China450 nm. 7 lysate(C,D) expressedSerum IgG1 at reciprocaland IgG2aendpoint responses titer. in vehicleOne-way only ANOVA or OMV-vaccinated was used to determine mice (PBS statistical n = 5, all significance, others n = ns 10)p > to 0.05, B. *malleip ≤ 0.05,China ** p7 ≤lysate0.01, expressed*** p ≤ 0.001, at ****reciprocalp ≤ 0.0001. endpoint titer. One-way ANOVA was used to determine statistical significance, ns p >0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Anti-B. mallei serum IgG1 and IgG2a levels were measured by determining the reciprocal Anti-B. mallei serum IgG1 and IgG2a levels were measured by determining the reciprocal end-point titer of reactivity to heat-killed B. mallei China 7 lysate (Figure4C,D). The IgG results end-point titer of reactivity to heat-killed B. mallei China 7 lysate (Figure 4C,D). The IgG results followed a similar trend as with the poly-Ig. TxDOH vaccinated mice had significantly higher amounts followed a similar trend as with the poly-Ig. TxDOH vaccinated mice had significantly higher of IgG1 and IgG2a in serum than all other vaccinated mice. The mean reciprocal IgG1 titer of E555 amounts of IgG1 and IgG2a in serum than all other vaccinated mice. The mean reciprocal IgG1 titer OMV vaccinated mice following the subcutaneous vaccination regimen was significantly higher than of E555 OMV vaccinated mice following the subcutaneous vaccination regimen was significantly Bp82 OMV or PBS vaccination. These values were used to calculate the IgG1/IgG2a ratio (Table1). higher than Bp82 OMV or PBS vaccination. These values were used to calculate the IgG1/IgG2a ratio If the ratio is below or close to one the antibody response can be more indicative of a cell mediated (Table 1). If the ratio is below or close to one the antibody response can be more indicative of a cell antibody response where IFN-γ suppresses IgG1 production and increases IgG2a production, while if mediated antibody response where IFN-γ suppresses IgG1 production and increases IgG2a much higher than 1, a type 2 antibody response predominates [63]. Vehicle only vaccinated mice have production, while if much higher than 1, a type 2 antibody response predominates [63]. Vehicle only no detectable IgG1 or IgG2. The order of ratio for OMV vaccinated mice is as follows TxDOH < Bp82 vaccinated mice have no detectable IgG1 or IgG2. The order of ratio for OMV vaccinated mice is as << E555, (4.09, 9.84, and 34.30, respectively). All ratios were above one, indicating a type 2 antibody follows TxDOH < Bp82 << E555, (4.09, 9.84, and 34.30, respectively). All ratios were above one, response is produced in mice following vaccination with all OMV preparations. indicating a type 2 antibody response is produced in mice following vaccination with all OMV preparations.

Vaccines 2018, 6, 5 11 of 20 Vaccines 2018, 6, 5 11 of 20

Table 1.1. OMVOMV immunizationsimmunizations induceinduce B.B. malleimallei speciﬁcspecific IgG1IgG1 andand IgG2aIgG2a responses.responses. Geometric Mean Titer 1 Antigen Group Geometric Mean Titer 1 Antigen Group IgG1 IgG2a IgG1/IgG2a Ratio IgG1 IgG2a IgG1/IgG2a Ratio PBS 100 100 1.00 Bp82PBS OMV 984 100 100 100 9.84 1.00 B. mallei China 7 lysate Bp82 OMV 984 100 9.84 B. mallei China 7 lysate E555 OMV 13718 400 34.30 E555 OMV 13718 400 34.30 TxDOHTxDOH OMV OMV 23702 23702 5796 5796 4.09 4.09 1 1 TheThe geometricgeometric mean mean of theof reciprocalthe reciprocal endpoint endpoint titers with titers the with IgG1/IgG2a the IgG1/IgG2a ratio of the ratio values of indicated the values for eachindicated group. for each group.

TheThe abilityability ofof thethe variousvarious serumserum samplessamples toto promotepromote opsonophagocyticopsonophagocytic uptakeuptake ofof B.B. malleimallei ChinaChina 77 byby murinemurine macrophagemacrophage cellcell lineline RAW264.7RAW264.7 waswas assessedassessed (Figure(Figure5 A).5A). Intracellular Intracellular bacteria bacteria numbersnumbers werewere enumeratedenumerated andand showedshowed thatthat pooledpooled andand heat-inactivatedheat-inactivated mousemouse serumserum fromfrom OMVOMV vaccinatedvaccinated micemice significantlysignificantly increasedincreased opsonophagocytosisopsonophagocytosis severalseveral magnitudesmagnitudes higherhigher thanthan pooledpooled PBSPBS vaccinated mouse mouse serum serum or or no no serum serum controls controls as asevidenced evidenced by the by theincrease increase in intracellular in intracellular CFU CFUfrom from~100 ~100 to several to several thousand thousand CFUs. CFUs. Calculated Calculated invasion invasion efficiencies efficiencies (intracellular(intracellular CFU/totalCFU/total inoculatedinoculated CFU)CFU) werewere as as follows; follows; No No serum serum and and PBS: PBS: 3.1 3.1× × 1010−−55, ,Bp82: Bp82: 8.2 8.2 ×× 1010−4,− E555:4, E555: 2.28 2.28 × 10×−310, and−3, andTxDOH: TxDOH: 1.60 1.60× 10−×3. Mouse10−3. Mouse serum serumfrom each from of each the three of the vaccinated three vaccinated mouse mousegroups groupswas also was used also to usedprobe to SDS-PAGE probe SDS-PAGE run cell runlysates cell from lysates five from B. mallei five B. isolates mallei isolatesin addition in addition to B. mallei to B. China mallei 7China (Figure 7 (Figure5B). Lanes5B). Lanes1–6 coincide 1–6 coincide with B. with malleiB. malleistrainsstrains China China7, G-2(3), 7, G-2(3), India India86-567-2, 86-567-2, Ivan, Ivan,SAVP1, SAVP1, and andTurkey Turkey #1; (obtained #1; (obtained from fromthe CDC). the CDC). Cell lysates Cell lysates were run were on runSDS-PAGE on SDS-PAGE followed followed by silver by staining silver stainingor Western or Westernblotting. blotting.Blotting the Blotting lysates the with lysates Bp82 with OMV Bp82 vaccinated OMV vaccinated mouse serum mouse shows serum the shows weak theinteraction weak interaction with the B. with mallei the lysates.B. mallei Thelysates. E555 and The TxDOH E555 and OMV TxDOH vaccinated OMV mouse vaccinated sera show mouse strong sera showreactivity strong to the reactivity LPS O-antigens to the LPS of O-antigensall isolates an ofd all even isolates to the and high even molecular to the high weight molecular CPS (indicated weight CPSby chevron). (indicated The by TxDOH chevron). OMV The vaccine TxDOH even OMV appears vaccine to even induce appears more toCPS induce reactive more antibodies CPS reactive than antibodiesthe E555 OMV than vaccine the E555 when OMV comparing vaccine when the Western comparing blots the between Western the blots two. between the two.

FigureFigure 5.5. OpsonophagocytosisOpsonophagocytosis of B.B. malleimallei byby RAW264.7RAW264.7 murinemurine macrophagesmacrophages andand serumserum reactivityreactivity toto otherother B.B. malleimallei strains.strains. ((AA)) NumberNumber ofof intracellularintracellular B.B. malleimallei followingfollowing opsonizationopsonization withwith OMV-vaccinatedOMV-vaccinated mousemouse serumserum byby RAW264.7RAW264.7 murinemurine macrophages.macrophages. SigniﬁcanceSignificance determineddetermined byby one-wayone-way ANOVA,ANOVA, * *p p≤ ≤0.05, 0.05, **** ****p p≤ ≤0.0001. 0.0001. ( B(B)) Silver Silver stain stain and and Western Western blots blots of reactivityof reactivity between between six differentsix differentB. mallei B. malleistrain strain lysates lysates and pooledand pooled mouse mouse serum serum from the from indicated the indicated OMV-vaccinated OMV-vaccinated mouse (nmouse = 10) (n groups. = 10) groups. Lanes 1–6, LanesB. mallei 1–6, strainsB. mallei China strains 7, G-2(3),China 7, India G-2(3), 86-567-2, India Ivan,86-567-2, SAVP1, Ivan, and SAVP1, Turkey and #1, respectively.Turkey #1, respectively. Caret indicates Caret high indicates molecular high weight molecular CPS weight reactivity. CPS reactivity.

3.4.3.4. AbilityAbility ofof OMVOMV Vaccines to ProtectProtect fromfrom AcuteAcute andand ChronicChronic InhalationalInhalational GlandersGlanders inin BALB/cBALB/c MiceMice TheThe abilityability ofof OMVsOMVs toto vaccinatevaccinate andand protectprotect micemice fromfrom lethallethal glandersglanders upup toto thethe criticalcritical 30-day30-day markmark waswas assessedassessed nextnext (Figure(Figure6 ).6). GroupsGroups of of 10 10 vehicle vehicle only only or or OMV-vaccinated OMV-vaccinated BALB/c BALB/c mice mice (prime(prime andand boostedboosted twicetwice atat two-weektwo-week intervals)intervals) werewere challengedchallenged 44 weeksweeks afterafter thethe ﬁnalfinal boostboost withwith an acutely lethal intranasal dose of 1.5 × 104 CFU B. mallei China 7 and survival proportions were recorded (Figure 6A). Mice receiving a mock vaccine of PBS succumbed within six days to acute

Vaccines 2018, 6, 5 12 of 20

Vaccines 2018, 6, 5 12 of 20 an acutely lethal intranasal dose of 1.5 × 104 CFU B. mallei China 7 and survival proportions were infection.recorded Two-thirds (Figure6A). of Mice Bp82 receiving OMV vaccinated a mock vaccine mice succumbed of PBS succumbed during the within same sixtime days period to acute with theinfection. remaining Two-thirds mice surviving of Bp82 out OMV to the vaccinated 35-day study, mice succumbedendpoint resulting during thein a samesignificant time period increase with in survivalthe remaining compared mice to surviving PBS vaccinated out to themice. 35-day Mice study,receiving endpoint either resultingthe E555 or in TxDOH a signiﬁcant OMV increase vaccines in weresurvival protected compared from to PBSacute vaccinated lethal glanders mice. Mice with receiving only eitherone TxDOH the E555 orOMV-vaccinated TxDOH OMV vaccinesmouse succumbingwere protected at 12 from days acute post-infection lethal glanders (dpi) with and only100% one of E555 TxDOH OMV-vaccinated OMV-vaccinated mice mouse surviving succumbing to the 35-dayat 12 days study post-infection endpoint. Statistical (dpi) and 100%significance of E555 of OMV-vaccinated the E555 and TxDOH mice surviving OMV vaccinated to the 35-day groups study comparedendpoint. to Statistical the vehicle signiﬁcance only group of thewith E555 the andnumber TxDOH of animals OMV vaccinated used has greater groups than compared 95% power to the butvehicle decreases only group to 50% with power the number when ofcomparing animals used the hasvehicle greater only than and 95% the power Bp82 but OMV decreases vaccinated to 50% groups.power when comparing the vehicle only and the Bp82 OMV vaccinated groups.

Survival of BALB/c IN challenged A with 1.5x104 CFU B. mallei China 7 100 **** Vaccine: 75 PBS ** Bp82 OMV 50 E555 OMV 25 *** TxDOH OMV

Percent survival 0 0 7 14 21 28 35 Days post challenge

Organ loads in BALB/c mice 35 days post-infection

7 B 3×10 C 1.5×106 D 2×108 1.0×106 1×108 2×107 5000 Vaccine: Bp82 OMV 4000 4×107 E555 OMV 1×107 3000 TxDOH OMV CFU/organ CFU/organ 2000 CFU/organ 7 1×106 2×10 2000 1000 1000 0 0 0 g n n er Lu v lee Li p S

FigureFigure 6. 6. OMVOMV vaccination vaccination protects protects BALB/c BALB/c mice mice from from acute acute lethal lethal glanders glanders following following intranasal intranasal inoculation.inoculation. (A) ( ASurvival) Survival to 35 to days 35 daysof 4–6 of weeks 4–6 weeksold BALB/ oldc BALB/cmice intranasally mice intranasally challenged challengedwith 1.5 × 4 10with CFU 1.5 B.× mallei104 CFUChinaB. 7 malleiafter prime-boost-boostChina 7 after prime-boost-boost vaccination with vaccination the Bp82-, E555-, with theTxDOH-derived Bp82-, E555-, OMVTxDOH-derived (each group OMV n = 10) (each or PBS group (n = n 5). = 10)Statistically or PBS (n significant = 5). Statistically increases signiﬁcant in survival increases compared in survival to PBS orcompared each other to PBSas indicated or each otherwere asdetermined indicated wereby the determined Log-rank (Mantel-Cox) by the Log-rank test (Mantel-Cox) where ** p ≤ test0.01, where *** p ≤** 0.001,p ≤ 0.01, **** p *** ≤ p0.0001.≤ 0.001, Bacterial **** p ≤loads0.0001. in the Bacterial lungs ( loadsB), livers in the (C lungs), and (spleensB), livers (D ()C of), and35-day spleens survivors. (D) of In35-day panels survivors. (B–D) each In panelssymbol ( Bequals–D) each one symbolmouse. equals one mouse.

However, mice appeared slightly emaciated so three Bp82 OMV, five E555 and four TxDOH However, mice appeared slightly emaciated so three Bp82 OMV, ﬁve E555 and four TxDOH mice were humanely sacrificed to evaluate bioburden in the lungs, livers, and spleens (Figure 6B–D). mice were humanely sacriﬁced to evaluate bioburden in the lungs, livers, and spleens (Figure6B–D). Organ bacterial burdens were determined and high CFU numbers were found in the spleens of all Organ bacterial burdens were determined and high CFU numbers were found in the spleens of survivors tested indicating these vaccines did not achieve effective immunity in BALB/c mice. Gross all survivors tested indicating these vaccines did not achieve effective immunity in BALB/c mice. pathology of spleens revealed numerous abscesses and splenomegaly. Each group had at least one Gross pathology of spleens revealed numerous abscesses and splenomegaly. Each group had at least mouse that had no detectable bacteria in the lungs. Three TxDOH-vaccinated mice and one one mouse that had no detectable bacteria in the lungs. Three TxDOH-vaccinated mice and one E555-vaccinated mouse had no detectable bacteria in their livers and one mouse from each had E555-vaccinated mouse had no detectable bacteria in their livers and one mouse from each had cleared cleared the bacteria in the lungs and livers. Perhaps this is due to high levels of neutralizing the bacteria in the lungs and livers. Perhaps this is due to high levels of neutralizing antibodies antibodies produced in the Th2 dominating immune response we measured in the BALB/c mouse produced in the Th2 dominating immune response we measured in the BALB/c mouse background background that then drives intracellular infections that occur in the spleen. that then drives intracellular infections that occur in the spleen. Protection beyond 35 days is also desirable because chronic infections can lead to death well beyond 35 days. Groups of 5 additional BALB/c mice receiving PBS, E555 OMV or TxDOH OMV vaccines were observed for survival following intranasal challenge with 1.5 × 104 CFU of B. mallei China 7 out to 60 dpi. Both vaccines afforded significant protection from acute lethal glanders to all

Vaccines 2018, 6, 5 13 of 20

Protection beyond 35 days is also desirable because chronic infections can lead to death well beyond 35 days. Groups of 5 additional BALB/c mice receiving PBS, E555 OMV or TxDOH OMV 4 vaccinesVaccines 2018 were, 6, 5 observed for survival following intranasal challenge with 1.5 × 10 CFU of B.13 mallei of 20 China 7 out to 60 dpi. Both vaccines afforded significant protection from acute lethal glanders to all mice and all all survived survived out out past past 30 30 dpi dpi (Figure (Figure 7A),7A), re reiteratingiterating the the findings findings in in Figure Figure 6A.6A. Starting Starting at at 43 43dpi, dpi, vaccinated vaccinated mice mice began began to succumb to succumb to chronic to chronic glanders glanders infection. infection. By the 60-day By the study 60-day endpoint, study endpoint,2/5 E555 OMV 2/5 E555 vaccinated OMV vaccinated mice survived mice survived and only and 1/5 only from 1/5 the from TxDOH the TxDOH vaccinated vaccinated group. group. The Theoverall overall increase increase in survival in survival in vaccinated in vaccinated groups groups was highly was highly significant significant compared compared to the to vehicle the vehicle only onlyvaccinated vaccinated controls. controls. Groups Groups of five of mice five mice gave gave 80% 80%power power to our to ourstatistical statistical significance significance at day at day 50 50post-infection. post-infection. Survivors Survivors were were humanely humanely sacrificed sacrificed and and bacterial bacterial organ organ loads loads were determineddetermined (Figure7 B).7B). Splenic Splenic loads loads were were very very high high in the in three the three survivors survivors with greater with greater than 1 ×than107 1CFU × 10 present.7 CFU Onepresent. E555 One OMV E555 vaccinated OMV vaccinated mouse had mouse no detectable had no detectable CFU in the CFU lung in the or liver lung and or liver the other and the also other had relativelyalso had relatively low levels. low Gross levels. pathology Gross ofpathology the organs of showed the organs chronic showed splenic chronic involvement splenic with involvement advanced abscesswith advanced formation, abscess substantial formation, splenomegaly, substantial and splenomegaly, tight splenic adherence and tight to splenic the intra-abdominal adherence to wall, the requiringintra-abdominal extensive wall, surgical requiring removal. extensive surgical removal.

Figure 7.7. OMVOMV vaccination vaccination protects BALB/c mice mice fr fromom chronic glanders following intranasal inoculation. ((AA)) 4–64–6 weeks weeks old old BALB/c BALB/c mice mice intranasally intranasally challenged challenged with with 1.5 × 1.5104 ×CFU 104 B.CFU mallei B. Chinamallei 7China after prime-boost-boost7 after prime-boost-boost vaccination vaccination with E555-, with TxDOH-derived E555-, TxDOH-derived OMV or PBSOMV (each or PBS group (each n = group 5) were n observed= 5) were observed for survival for outsurvival to 60 out days. to 60 Statistically days. Statistically signiﬁcant significant increases increases in survival in survival compared compared to PBS vaccinatedto PBS vaccinated mice were mice determined were determined by the Log-rank by the (Mantel-Cox)Log-rank (Mantel-Cox) test where **testp ≤where0.01. The** p red-dashed≤ 0.01. The linered-dashed indicates line an indicates important an milestone important of milestone 30 days post-infection. of 30 days post-infection. (B) Bacterial ( loadsB) Bacterial in the lungs,loads livers,in the andlungs, spleens livers, of and 60-day spleens survivors. of 60-day survivors.

3.5. Ability of B. thailandensis E555 OMV Vaccine to Protect from AcuteAcute Lethal Glanders in C57BL/6 Mice The OMVOMV vaccinevaccine produced produced from fromB. thailandensisB. thailandensisE555 E555 was was chosen chosen as the as best the candidatebest candidate to assess to inassess the well-characterizedin the well-characterized C57BL/6 C57BL/6 mouse mouse because because of the of high the levelshigh levels of antibodies of antibodies specific specific to both to B.both mallei B. Chinamallei 7China and B. 7 pseudomallei and B. pseudomallei1026b lysates 1026b (Figure lysates4). Five (Figure C57BL/6 4). miceFive wereC57BL/6 vaccinated mice withwere E555vaccinated OMVs with using E555 the sameOMVs regimen using the as insame the studiesregimen with as BALB/cin the studies mice, thenwith challengedBALB/c mice, with then the samechallenged acutely with lethal the dose same of acutelyB. mallei lethalChina dose 7 (17 of× B.LD mallei50 in China C57BL/6 7 (17 mice, × LD and50 in observed C57BL/6 for mice, survival and (Figureobserved8A). for All survival PBS vaccinated (Figure control 8A). All mice PBS died vaccinated by 4 dpi. E555control OMV mice vaccination died by provided 4 dpi. E555 significant OMV protectionvaccination from provided acute lethalsignificant glanders. protection One mouse from acute succumbed lethal toglanders. acute glanders One mouse on day succumbed 3. A second to mouseacute glanders with neurological on day 3. symptomsA second thatmouse otherwise with neurological appeared healthy symptoms was that humanely otherwise euthanized appeared at 10healthy dpi. was The otherhumanely three euthanized mice survived at 10 to dpi. day The 21, other the study three endpoint. mice survived The threeto day survivors 21, the study were sacrificedendpoint. andThe bacterial three survivors burden was were assessed sacrificed by organ and loadbacterial determination. burden was Figure assessed8B shows by thatorgan mouse load 1determination. had ~250 CFU Figure of B. mallei 8B showsin the that lung mouse and had 1 had no measurable ~250 CFU bacterialof B. mallei CFU in inthe the lung livers and or had spleen. no Themeasurable other two bacterial mice hadCFU no in measurable the livers orB. sp malleileen. CFUThe other in lungs, two livers,mice had or spleens,no measurable thus indicating B. mallei efficientCFU in lungs, bacterial livers, clearance or spleens, was achieved thus indicating in 40% ofefficient mice (2/5), bacterial and nearlyclearance achieved was achieved in the third in 40% mouse of mice (2/5), and nearly achieved in the third mouse that had bacteria detected in the lungs but not the liver or spleen. Establishment of a stronger Th1 response in the C57BL/6 background could explain why the surviving mice had cleared most of the bacteria. C57BL/6 mice are more resistant to infection resulting in a lower LD50 equivalency challenge dose compared to the BALB/c mice but was still well within a high-dose LD100 range. Future studies will look at the T-cell responses to OMV vaccination in the C57BL/6 mice.

Vaccines 2018, 6, 5 14 of 20 that had bacteria detected in the lungs but not the liver or spleen. Establishment of a stronger Th1 response in the C57BL/6 background could explain why the surviving mice had cleared most of the bacteria. C57BL/6 mice are more resistant to infection resulting in a lower LD50 equivalency challenge dose compared to the BALB/c mice but was still well within a high-dose LD100 range. Future studies Vaccineswill look 2018 at, 6 the, 5 T-cell responses to OMV vaccination in the C57BL/6 mice. 14 of 20

A Survival of C57BL/6 mice IN challenged B Organ loads in 21 dpi C57BL/6 survivors with 1.5x104 CFU B. mallei China 7 Mouse 1 Mouse 2 Mouse 3 300 100 Vaccination 75 * PBS 200 E555 OMV 50 100 CFU/organ 25

Percent survival Percent 0 0 0 7 14 21 g n n n n ng e ver u lee u iver e i Day post-infection L Liverp L L pl LungL plee S S S

FigureFigure 8. OMVs derivedderived fromfromB. B. thailandensis thailandensisE555 E555 protect protect C57BL/6 C57BL/6 mice mice from from acute acute lethal lethal glanders glanders and andcan providecan provide sterilizing sterilizing immunity immunity following following intranasal intranasal inoculation. inoculation. (A) 4–6 (A) weeks 4–6 weeks old C57BL/6 old C57BL/6 mice miceintranasally intranasally challenged challenged with 1.5with× 1.5104 ×CFU 104 CFUB. mallei B. malleiChina China 7 after 7 after prime-boost-boost prime-boost-boost vaccination vaccination with withE555-derived E555-derived OMV OMV or PBS or (each PBS group(each ngroup = 5) were n = observed5) were observed for survival for out survival to 21 days. out to Statistically 21 days. Statisticallysigniﬁcant increasessignificant in increases survival comparedin survival to comp PBS vaccinatedared to PBS mice vaccinated were determined mice were bydetermined the Log-rank by the(Mantel-Cox) Log-rank test(Mantel-Cox) where * p ≤ test0.05. where Arrow * indicates p ≤ 0.05. mouse Arrow was indicates euthanized mouse for neurological was euthanized symptoms. for neurological(B) Organ loads symptoms. of lungs, ( livers,B) Organ and loads spleens of oflungs, 21-day livers, survivors and spleens showing of sterilizing21-day survivors immunity showing in two sterilizingmice and closeimmunity to sterilizing in two mice immunity and clos in thee to third.sterilizing immunity in the third.

4.4. Discussion Discussion B.B. mallei mallei isis a a lesser lesser known known but but no no less less no notorioustorious biological biological warfare warfare agent. agent. The The disease disease it it causes causes is is difficultdifficult toto prevent prevent due due to high to pathogenicityhigh pathogenicity and the and intracellular the intracellular nature of thenature organism. of the Development organism. Developmentof a vaccine for of glanders a vaccine has for been glanders focused has mainly been fo oncused subunit mainly vaccines on subunit that have vaccines had varying that have degrees had varyingof effectiveness degrees [35of ,36effectiveness,64] and live-attenuated [35,36,64] and vaccines live-attenuated [14,33,34 vaccines] that face [14,33,34] many hurdles that face to human many hurdlesuse. Through to human our useuse. of Through biosafe strain-derived our use of biosaf OMVse strain-derived to generate protective OMVs to immune generate responses protective we immunehave circumvented responses we both have issues. circumvented We showed both that issues. the OMVsWe showed from allthat strains the OMVs have from CPS associatedall strains havewith them.CPS associated Bp82 OMVs with generatedthem. Bp82 very OMVs poor generated anti-B. mallei veryantibody poor anti- levelsB. mallei compared antibody to levels either comparedB. thailandensis to either-derived B. thailandensis- vaccine andderived could vaccine explain and why could the Bp82 explain OMVs why provided the Bp82 significantlyOMVs provided less significantlyprotection. Theless successprotection. of other The OMVsuccess vaccines, of other including OMV vaccines, the melioidosis including OMV the melioidosis vaccine, has OMV been vaccine,attributed has to been strong attributed type 2 antibody to strong responses type 2 anti [44body,65,66 ].responses In this study, [44,65,66]. strong In antibody this study, responses strong antibodywere observed responses following were observed vaccination following with OMVs vaccination particularly with towardsOMVs particularly the LPS O-antigen towards andthe CPS.LPS O-antigenOther works and haveCPS. shownOther works high IgGhave and shown IgM high responses IgG and are IgM generated responses by are mice generated vaccinated by mice with vaccinatedthe CPS from withB. the thailandensis CPS from E555B. thailandensis [30]. The E555 higher [30]. immunogenicity The higher immunogenicity of lipid A in ofB. lipid thailandensis A in B. thailandensisLPS may contribute LPS may to bettercontribute memory to better responses memory by inducing responses stronger by inducing local inflammation stronger local that inflammationattracts more antigenthat attracts presenting more cells.antigen Lipid presen A andting liposomescells. Lipid containing A and liposomes immunogenic containing lipid A immunogenicare known to belipid potent A are adjuvants known to for be vaccines potent adjuvants and help inducefor vaccines Th1 responses and help [induce67,68]. Th1 Figure responses A2 also [67,68].demonstrated Figure thatA2 also the proteindemonstrated content that of thetheOMVs protein vary content substantially. of the OMVs It appears vary substantially. that the OMVs It appearsfrom B. thailandensisthat the OMVscontain from higher B. thailandensis quantities ofcontain common higher proteins quantities and several of common proteins proteins that are and not severalfound inproteinsB. pseudomallei that are -derivednot found OMVs. in B. pseudomallei These may-derived have been OMVs. protective These may antigens have that been generated protective a antigenscell-mediated that generated response leading a cell-mediated to protection response in the vaccinationleading to modelprotection utilized. in the The vaccination identities ofmodel these utilized.proteins The will identities be determined of these in futureproteins analysis. will be determined in future analysis. TheThe BALB/c BALB/c mouse mouse model model is is generally generally accepted accepted to to be be an an appropriate appropriate and and moderately moderately sensitive sensitive infectiousinfectious disease disease model model for for melioidosis melioidosis and and glanders glanders [69,70]. [69,70]. Despite Despite this this we we were were able able to to elicit elicit significant protection to acute lethal challenge of 22 × LD50 and even to chronic lethal glanders. However, immunity was not sterilizing and protection from chronic glanders was incomplete; potentially due to the enhanced type 2 antibody responses that dominate the BALB/c mouse immune response [71–74]. Chronically infected BALB/c survivors had nearly cleared bacteria from the lungs but the high burden in the spleen indicated a failure of proper cell-mediated immune responses in this model. Since C57BL/6 mice are generally more resistant to B. mallei infection and generate a more Th1-skewed response we wanted to test our best vaccine candidate in this inbred mouse line. The decision to move forward with E555 OMVs was two-fold: 1. It was able to generate

Vaccines 2018, 6, 5 15 of 20

significant protection to acute lethal challenge of 22 × LD50 and even to chronic lethal glanders. However, immunity was not sterilizing and protection from chronic glanders was incomplete; potentially due to the enhanced type 2 antibody responses that dominate the BALB/c mouse immune response [71–74]. Chronically infected BALB/c survivors had nearly cleared bacteria from the lungs but the high burden in the spleen indicated a failure of proper cell-mediated immune responses in this model. Since C57BL/6 mice are generally more resistant to B. mallei infection and generate a more Th1-skewed response we wanted to test our best vaccine candidate in this inbred mouse line. The decision to move forward with E555 OMVs was two-fold: 1. It was able to generate high amounts of antibodies to both B. mallei and B. pseudomallei lysate. 2. It provided 100% protection from acute lethal glanders in BALB/c mice. Testing in C57BL/6 mice showed significant protection from acute lethal glanders and 2/5 mice had no measurable bacteria in the target organs. The livers and spleens of 3/5 mice were free of detectable bacteria. The OMV vaccines generated in this work are excellent candidates for a human vaccine. They are safe, easy, and cheap to produce. These strains are not considered human pathogens so infectivity is not a worry and biosafety concerns associated with growth volume are considerably reduced. The one-liter growth preparation used in this study typically results in 100–200 mg of lyophilized OMV material. For perspectives sake, that is enough material to vaccinate ~13,000–26,000 mice with the full three-dose vaccine regimen. Human doses are surely higher but even then, tangential flow filtration can be added to the workflow for high volume scale-up if desired. Another bonus is stability. The OMVs can be lyophilized, frozen, thawed, and rehydrated while retaining their immunogenic properties (this work) and can also provide a protective environment for active enzymes [75]. Future directions include increasing the dose of OMV vaccine given. Our dose of 2.5 µg is a modest dose and could be increased to see if protection from a higher dose challenge can be achieved. We also found a sizeable portion of the antibody response was to the LPS O-antigen. It is possible that the numerous repetitive T-cell independent antigens present in LPS are masking protein antigens important for generating cell-mediated immune responses. Eliminating these components and observing the effect on antibody production or survival in mice is another way we can enhance effective T-cell memory responses. Future work in this direction would lead to greater understanding of protective cell-mediated Th1 responses generated in both BALB/c and C57BL/6 glanders models. We would also like to test its potential as a divalent vaccine by assessing ability to prevent acute lethal melioidosis in addition to glanders. Since high levels of anti-B. pseudomallei antibodies were observed, the E555 OMV vaccine could theoretically be used to vaccinate against both glanders and melioidosis. Vaccine formulations in this work did not include any type of licensed approved adjuvant or material beyond what is present in the PBS OMV suspension. Future formulations incorporating vaccine adjuvants such as CpG or poly(I:C) for stimulation of Th1 responses could lead to a higher likelihood of complete immunity.

5. Conclusions In conclusion, we believe this is a step forward in vaccine development against glanders and for many of the reasons described above, biosafe strain-derived OMVs represent good candidates for targeted research and development.

Acknowledgments: We thank Direk Limmathurotsakul, Mahidol University, Thailand for providing B. thailandensis strain E555; and Alex R. Hoffmaster, CDC Atlanta, for providing B. thailandensis TxDOH. We would like to thank Narisara Chantratita of Mahidol University for the kind gift of the latex agglutination test and Daniel Sanford of Battelle for the gift of the non-human primate serum. mAbs 4C7 and 4C4 were kindly provided by David AuCoin of the University of Nevada, Reno. This work was supported by University of Florida’s startup funds to A.T. and the UF Emerging Pathogens Institute Seed grant 16-3 to M.H.N. Author Contributions: M.H.N. and A.T. conceived and designed the experiments; M.H.N., M.S.R.K, S.C. and A.T. performed the experiments; M.H.N. and A.T. analyzed the data; H.P.S. contributed reagents and materials; M.H.N. wrote the paper. All authors reviewed the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Vaccines 2018, 6, 5 16 of 20

Vaccines 2018, 6, 5 16 of 20 Vaccines 2018, 6, 5 16 of 20 Appendix A AppendixAppendix A A

FigureFigureFigure A1. A1.A1. Non-human Non-humanNon-human primate primateprimate pre/post pre/postpre/post challenge challenge anti-LPS anti-LPS responses. responses.

Figure A2. FigureFigure A2. A2. Coomassie Coomassie stains stains of of OMV OMV preparations preparations compared compared to toto cell cellcell lysates. lysates.lysates.

ReferencesReferences

1.1. Sharrer,Sharrer, G.T. G.T. The The great great great glanders glanders glanders epizootic, epizootic, epizootic, 1861–1866: 1861–1866: 1861–1866: A A civil Acivil civil war war warlegacy. legacy. legacy. Agric. Agric.Agric. Hist. Hist. 1995 Hist.1995, ,69 199569, ,79–97. 79–97., 69, 79–97.

2.2. Galyov,[Galyov,PubMed E.E.; E.E.;] Brett, Brett, P.J.; P.J.; DeShazer, DeShazer, D. D. Molecular Molecular insights insights into into burkholderia burkholderia pseudomallei pseudomallei and and burkholderia burkholderia 2. malleiGalyov,mallei pathogenesis. pathogenesis. E.E.; Brett, P.J.;Annu. Annu. DeShazer, Rev. Rev. Microbiol. Microbiol. D. Molecular 2010 2010, ,64 insights64, ,495–517. 495–517. into Burkholderia pseudomallei and Burkholderia mallei

3.3. Khan,pathogenesis.Khan, I.; I.; Wieler, Wieler,Annu. L.H.; L.H.; Rev. Melzer, Melzer, Microbiol. F.; F.; Elschner, Elschner,2010, 64 ,M.C.; 495–517.M.C.; Muhammad, Muhammad, [CrossRef ][G.; G.;PubMed Ali, Ali, S.; S.;] Sprague, Sprague, L.D.; L.D.; Neubauer, Neubauer, H.; H.; 3. Saqib,Khan,Saqib, M. I.;M. Wieler, GlandersGlanders L.H.; ininMelzer, animals:animals: F.; Elschner,AA reviewreview M.C.; onon ep Muhammad,epidemiology,idemiology, G.; clinicalclinical Ali, S.; presentation, Sprague,presentation, L.D.; diagnosisdiagnosis Neubauer, andand H.; countermeasures.Saqib,countermeasures. M. Glanders Transbound. Transbound. in animals: Emerg. Emerg. ADis. Dis. review 2013 2013, ,60 on60, ,204–221. 204–221. epidemiology, clinical presentation, diagnosis and

4.4. Vancountermeasures.Van Zandt, Zandt, K.E.; K.E.; Greer, Greer,Transbound. M.T.; M.T.; Gelhaus, Emerg.Gelhaus, Dis. H.C. H.C.2013 Gl Gl,anders:60anders:, 204–221. An An overview [overviewCrossRef of ][of PubMedinfection infection] in in humans. humans. Orphanet Orphanet J. J. 4. RareVanRare Dis. Zandt,Dis. 2013 2013 K.E.;, ,8 8, ,doi:10.1186/1750-1172-8-131. doi:10.1186/1750-1172-8-131. Greer, M.T.; Gelhaus, H.C. Glanders: An overview of infection in humans. Orphanet J.

5.5. Wheelis,RareWheelis, Dis. M. 2013M. First First, 8 .[shots shotsCrossRef fire firedd][ in inPubMed biological biological] warfare. warfare. Nature Nature 1998 1998, ,395 395, ,doi:10.1038/26089. doi:10.1038/26089.

6.5.6. Dance,Wheelis,Dance, D.A. D.A. M. Melioidosis FirstMelioidosis shots ﬁredand and glanders in glanders biological as as possible warfare.possible biological biologicalNature 1998 weapons. weapons., 395.[CrossRef In In Bioterrorism Bioterrorism][PubMed and and ]Infectious Infectious Agents Agents; ; Alibek,Alibek, F.A., F.A., Ed.; Ed.; Springer Springer Sc Science+Businessience+Business Media Media Inc.: Inc.: New New York, York, NY, NY, USA, USA, 2005; 2005; pp. pp. 99–145. 99–145.

7.7. Srinivasan,Srinivasan, A.; A.; Kraus, Kraus, C.N.; C.N.; DeShazer, DeShazer, D.; D.; Becker, Becker, P.M.; P.M.; Dick, Dick, J.D.; J.D.; Spacek, Spacek, L.; L.; Bartlett, Bartlett, J.G.; J.G.; Byrne, Byrne, W.R.; W.R.; Thomas,Thomas, D.L. D.L. Glanders Glanders in in a am militaryilitary research research microbiologist. microbiologist. N. N. Engl. Engl. J. J. Med. Med. 2001 2001, ,345 345, ,256 256––258.258.

Vaccines 2018, 6, 5 17 of 20

6. Dance, D.A. Melioidosis and glanders as possible biological weapons. In Bioterrorism and Infectious Agents; Alibek, F.A., Ed.; Springer Science+Business Media Inc.: New York, NY, USA, 2005; pp. 99–145. 7. Srinivasan, A.; Kraus, C.N.; DeShazer, D.; Becker, P.M.; Dick, J.D.; Spacek, L.; Bartlett, J.G.; Byrne, W.R.; Thomas, D.L. Glanders in a military research microbiologist. N. Engl. J. Med. 2001, 345, 256–258. [CrossRef] [PubMed] 8. Miller, W.R.; Pannell, L.; Cravitz, L.; Tanner, W.A.; Ingalls, M.S. Studies on certain biological characteristics of malleomyces mallei and malleomyces pseudomallei: I. Morphology, cultivation, viability, and isolation from contaminated specimens. J. Bacteriol. 1948, 55, 115–126. [PubMed] 9. Miller, W.R.; Pannell, L.; Cravitz, L.; Tanner, W.A.; Rosebury, T. Studies on certain biological characteristics of malleomyces mallei and malleomyces pseudomallei: II. Virulence and infectivity for animals. J. Bacteriol. 1948, 55, 127–135. [PubMed] 10. Wittig, M.B.; Wohlsein, P.; Hagen, R.M.; Al Dahouk, S.; Tomaso, H.; Scholz, H.C.; Nikolaou, K.; Wernery, R.; Wernery, U.; Kinne, J.; et al. Glanders—A comprehensive review. DTW. Dtsch. Tierarztliche Wochenschr. 2006, 113, 323–330. 11. Withers, M.R. Bacterial agents: Glanders and melioidosis. In Usamriid’s Medical Management of Biological Casualties Handbook, 8th ed.; U.S. Army Medical Research Institute of Infectious Diseases: Frederick, MD, USA, 2014; pp. 51–58. 12. Goodyear, A.; Kellihan, L.; Bielefeldt-Ohmann, H.; Troyer, R.; Propst, K.; Dow, S. Protection from pneumonic infection with burkholderia species by inhalational immunotherapy. Infect. Immun. 2009, 77, 1579–1588. [CrossRef][PubMed] 13. Massey, S.; Johnston, K.; Mott, T.M.; Judy, B.M.; Kvitko, B.H.; Schweizer, H.P.; Estes, D.M.; Torres, A.G. In vivo bioluminescence imaging of Burkholderia mallei respiratory infection and treatment in the mouse model. Front. Microbiol. 2011, 2.[CrossRef][PubMed] 14. Mott, T.M.; Vijayakumar, S.; Sbrana, E.; Endsley, J.J.; Torres, A.G. Characterization of the Burkholderia mallei tonb mutant and its potential as a backbone strain for vaccine development. PLoS Negl. Trop. Dis. 2015, 9, e0003863. [CrossRef][PubMed] 15. Nierman, W.C.; DeShazer, D.; Kim, H.S.; Tettelin, H.; Nelson, K.E.; Feldblyum, T.; Ulrich, R.L.; Ronning, C.M.; Brinkac, L.M.; Daugherty, S.C.; et al. Structural ﬂexibility in the Burkholderia mallei genome. Proc. Natl. Acad. Sci. USA 2004, 101, 14246–14251. [CrossRef][PubMed] 16. Ong, C.; Ooi, C.H.; Wang, D.; Chong, H.; Ng, K.C.; Rodrigues, F.; Lee, M.A.; Tan, P. Patterns of large-scale genomic variation in virulent and avirulent burkholderia species. Genome Res. 2004, 14, 2295–2307. [CrossRef] [PubMed] 17. Brett, P.J.; Deshazer, D.; Woods, D.E. Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains. Epidemiol. Infect. 1997, 118, 137–148. [CrossRef][PubMed] 18. Deshazer, D. Virulence of clinical and environmental isolates of burkholderia oklahomensis and Burkholderia thailandensis in hamsters and mice. FEMS Microbiol. Lett. 2007, 277, 64–69. [CrossRef][PubMed] 19. Haraga, A.; West, T.E.; Brittnacher, M.J.; Skerrett, S.J.; Miller, S.I. Burkholderia thailandensis as a model system for the study of the virulence-associated type iii secretion system of Burkholderia pseudomallei. Infect. Immun. 2008, 76, 5402–5411. [CrossRef][PubMed] 20. Schell, M.A.; Ulrich, R.L.; Ribot, W.J.; Brueggemann, E.E.; Hines, H.B.; Chen, D.; Lipscomb, L.; Kim, H.S.; Mrazek, J.; Nierman, W.C.; et al. Type vi secretion is a major virulence determinant in Burkholderia mallei. Mol. Microbiol. 2007, 64, 1466–1485. [CrossRef][PubMed] 21. Burtnick, M.N.; Brett, P.J.; Harding, S.V.; Ngugi, S.A.; Ribot, W.J.; Chantratita, N.; Scorpio, A.; Milne, T.S.; Dean, R.E.; Fritz, D.L.; et al. The cluster 1 type vi secretion system is a major virulence determinant in Burkholderia pseudomallei. Infect. Immun. 2011, 79, 1512–1525. [CrossRef][PubMed] 22. Schwarz, S.; Singh, P.; Robertson, J.D.; LeRoux, M.; Skerrett, S.J.; Goodlett, D.R.; West, T.E.; Mougous, J.D. Vgrg-5 is a burkholderia type vi secretion system-exported protein required for multinucleated giant cell formation and virulence. Infect. Immun. 2014, 82, 1445–1452. [CrossRef][PubMed] 23. Stevens, J.M.; Ulrich, R.L.; Taylor, L.A.; Wood, M.W.; Deshazer, D.; Stevens, M.P.; Galyov, E.E. Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bima mutant. J. Bacteriol. 2005, 187, 7857–7862. [CrossRef] [PubMed] Vaccines 2018, 6, 5 18 of 20

24. Stone, J.K.; Mayo, M.; Grasso, S.A.; Ginther, J.L.; Warrington, S.D.; Allender, C.J.; Doyle, A.; Georgia, S.; Kaestli, M.; Broomall, S.M.; et al. Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species. BMC Microbiol. 2012, 12.[CrossRef][PubMed] 25. Qazi, O.; Prior, J.L.; Judy, B.M.; Whitlock, G.C.; Kitto, G.B.; Torres, A.G.; Estes, D.M.; Brown, K.A. Sero-characterization of lipopolysaccharide from Burkholderia thailandensis. Trans. R. Soc. Trop. Med. Hyg. 2008, 102, S58–S60. [CrossRef] 26. Brett, P.J.; Burtnick, M.N.; Heiss, C.; Azadi, P.; DeShazer, D.; Woods, D.E.; Gherardini, F.C. Burkholderia thailandensis oaca mutants facilitate the expression of Burkholderia mallei-like o polysaccharides. Infect. Immun. 2011, 79, 961–969. [CrossRef][PubMed] 27. Tuanyok, A.; Stone, J.K.; Mayo, M.; Kaestli, M.; Gruendike, J.; Georgia, S.; Warrington, S.; Mullins, T.; Allender, C.J.; Wagner, D.M.; et al. The genetic and molecular basis of O-antigenic diversity in Burkholderia pseudomallei lipopolysaccharide. PLoS Negl. Trop. Dis. 2012, 6, e1453. [CrossRef][PubMed] 28. Reckseidler-Zenteno, S.L.; De Vinney, R.; Woods, D.E. The capsular polysacharride of Burkholderia pseudomallei contributes to survival in the serum by reducing complement factor c3b deposition. Infect. Immun. 2005, 73, 1106–1115. [CrossRef][PubMed] 29. Sim, B.M.; Chantratita, N.; Ooi, W.F.; Nandi, T.; Tewhey, R.; Wuthiekanun, V.; Thaipadungpanit, J.; Tumapa, S.; Ariyaratne, P.; Sung, W.K.; et al. Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic burkholderia isolates. Genome Biol. 2010, 11.[CrossRef][PubMed] 30. Bayliss, M.; Donaldson, M.I.; Nepogodiev, S.A.; Pergolizzi, G.; Scott, A.E.; Harmer, N.J.; Field, R.A.; Prior, J.L. Structural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain e555:: Wbii (pknock-kmr) and assessment of the signiﬁcance of the 2-O-acetyl group in immune protection. Carbohydr. Res. 2017, 452, 17–24. [CrossRef][PubMed] 31. Scott, A.E.; Laws, T.R.; D’Elia, R.V.; Stokes, M.G.; Nandi, T.; Williamson, E.D.; Tan, P.; Prior, J.L.; Atkins, T.P. Protection against experimental melioidosis following immunization with live Burkholderia thailandensis expressing a manno-heptose capsule. Clin. Vaccine Immunol. CVI 2013, 20, 1041–1047. [CrossRef][PubMed] 32. Moustafa, D.A.; Scarff, J.M.; Garcia, P.P.; Cassidy, S.K.; DiGiandomenico, A.; Waag, D.M.; Inzana, T.J.; Goldberg, J.B. Recombinant salmonella expressing Burkholderia mallei lps o antigen provides protection in a murine model of melioidosis and glanders. PLoS ONE 2015, 10, e0132032. [CrossRef][PubMed] 33. Hatcher, C.L.; Mott, T.M.; Muruato, L.A.; Sbrana, E.; Torres, A.G. Burkholderia mallei clh001 attenuated vaccine strain is immunogenic and protects against acute respiratory glanders. Infect. Immun. 2016, 84, 2345–2354. [CrossRef][PubMed] 34. Ulrich, R.L.; Amemiya, K.; Waag, D.M.; Roy, C.J.; DeShazer, D. Aerogenic vaccination with a Burkholderia mallei auxothroph protects against aerosol-initiated glanders in mice. Vaccine 2005, 23, 1986–1992. [CrossRef][PubMed] 35. Gregory, A.E.; Judy, B.M.; Qazi, O.; Blumentritt, C.A.; Brown, K.A.; Shaw, A.M.; Torres, A.G.; Titball, R.W. A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei. Nanomed. Nanotechnol. Biol. Med. 2015, 11, 447–456. [CrossRef][PubMed] 36. Torres, A.G.; Gregory, A.E.; Hatcher, C.L.; Vinet-Oliphant, H.; Morici, L.A.; Titball, R.W.; Roy, C.J. Protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine. Vaccine 2015, 33, 686–692. [CrossRef][PubMed] 37. Scott, A.E.; Burtnick, M.N.; Stokes, M.G.; Whelan, A.O.; Williamson, E.D.; Atkins, T.P.; Prior, J.L.; Brett, P.J. Burkholderia pseudomallei capsular polysaccharide conjugates provide protection against acute melioidosis. Infect. Immun. 2014, 82, 3206–3213. [CrossRef][PubMed] 38. Burtnick, M.N.; Heiss, C.; Roberts, R.A.; Schweizer, H.P.; Azadi, P.; Brett, P.J. Development of capsular polysaccharide-based glycoconjugates for immunization against melioidosis and glanders. Front. Cell. Infect. Microbiol. 2012, 2.[CrossRef][PubMed] 39. McBroom, A.J.; Kuehn, M.J. Release of outer membrane vesicles by gram-negative bacteria is a novel envelope stress response. Mol. Microbiol. 2007, 63, 545–558. [CrossRef][PubMed] 40. Choi, D.S.; Kim, D.K.; Choi, S.J.; Lee, J.; Choi, J.P.; Rho, S.; Park, S.H.; Kim, Y.K.; Hwang, D.; Gho, Y.S. Proteomic analysis of outer membrane vesicles derived from pseudomonas aeruginosa. Proteomics 2011, 11, 3424–3429. [CrossRef][PubMed] Vaccines 2018, 6, 5 19 of 20

41. Lee, E.Y.; Bang, J.Y.; Park, G.W.; Choi, D.S.; Kang, J.S.; Kim, H.J.; Park, K.S.; Lee, J.O.; Kim, Y.K.; Kwon, K.H.; et al. Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli. Proteomics 2007, 7, 3143–3153. [CrossRef][PubMed] 42. Van der Pol, L.; Stork, M.; van der Ley, P. Outer membrane vesicles as platform vaccine technology. Biotechnol. J. 2015, 10, 1689–1706. [CrossRef][PubMed] 43. Nieves, W.; Petersen, H.; Judy, B.M.; Blumentritt, C.A.; Russell-Lodrigue, K.; Roy, C.J.; Torres, A.G.; Morici, L.A. A Burkholderia pseudomallei outer membrane vesicle vaccine provides protection against lethal sepsis. Clin. Vaccine Immunol. CVI 2014, 21, 747–754. [CrossRef][PubMed] 44. Nieves, W.; Asakrah, S.; Qazi, O.; Brown, K.A.; Kurtz, J.; Aucoin, D.P.; McLachlan, J.B.; Roy, C.J.; Morici, L.A. A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection. Vaccine 2011, 29, 8381–8389. [CrossRef][PubMed] 45. Propst, K.L.; Mima, T.; Choi, K.H.; Dow, S.W.; Schweizer, H.P. A Burkholderia pseudomallei δpurm mutant is avirulent in immunocompetent and immunodeficient animals: Candidate strain for exclusion from select-agent lists. Infect. Immun. 2010, 78, 3136–3143. [CrossRef][PubMed] 46. Norris, M.H.; Kang, Y.; Lu, D.; Wilcox, B.A.; Hoang, T.T. Glyphosate resistance as a novel select-agent-compliant, non-antibiotic selectable-marker in chromosomal mutagenesis of the essential genes asd and dapb of Burkholderia pseudomallei. Appl. Environ. Microbiol. 2009, 75, 6062–6075. [CrossRef][PubMed] 47. Norris, M.H.; Kang, Y.; Wilcox, B.; Hoang, T.T. Stable, site-specific fluorescent tagging constructs optimized for burkholderia species. Appl. Environ. Microbiol. 2010, 76, 7635–7640. [CrossRef][PubMed] 48. Norris, M.H.; Propst, K.L.; Kang, Y.; Dow, S.W.; Schweizer, H.P.; Hoang, T.T. The Burkholderia pseudomallei δasd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice. Infect. Immun. 2011, 79, 4010–4018. [CrossRef][PubMed] 49. Norris, M.H. Identifying Virulence Factors and Regulators Contributing to Pathogenesis by the Select-Agent Bacterium Burkholderia pseudomallei. Docotral Thesis, University of Hawaii at Manoa, Honolulu, HI, USA, 2014. 50. Norris, M.H.; Rahman Khan, M.S.; Schweizer, H.P.; Tuanyok, A. An avirulent Burkholderia pseudomallei ∆purm strain with atypical type b lps: Expansion of the toolkit for biosafe studies of melioidosis. BMC Microbiol. 2017, 17, 132. [CrossRef][PubMed] 51. Norris, M.H.; Schweizer, H.P.; Tuanyok, A. Structural diversity of Burkholderia pseudomallei lipopolysaccharides affects innate immune signaling. PLoS Negl. Trop. Dis. 2017, 11, e0005571. [CrossRef] [PubMed] 52. Kulp, A.; Kuehn, M.J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 2010, 64, 163–184. [CrossRef][PubMed] 53. Lam, J.S.; Anderson, E.M.; Hao, Y. Lps quantitation procedures. Methods Mol. Biol. 2014, 1149, 375–402. [PubMed] 54. Lindner, B. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipopolysaccharides. In Bacterial Toxins: Methods and Protocols; Holst, O., Ed.; Humana Press: Totowa, NJ, USA, 2000; pp. 311–325. 55. Brett, P.J.; Burtnick, M.N.; Snyder, D.S.; Shannon, J.G.; Azadi, P.; Gherardini, F.C. Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human toll-like receptor 4 complexes. Mol. Microbiol. 2007, 63, 379–390. [CrossRef][PubMed] 56. Goodyear, A.; Troyer, R.; Bielefeldt-Ohmann, H.; Dow, S. Myd88-dependent recruitment of monocytes and dendritic cells required for protection from pulmonary Burkholderia mallei infection. Infect. Immun. 2012, 80, 110–120. [CrossRef][PubMed] 57. Anuntagool, N.; Naigowit, P.; Petkanchanapong, V.; Aramsri, P.; Panichakul, T.; Sirisinha, S. Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. J. Med. Microbiol. 2000, 49, 1075–1078. [CrossRef][PubMed] 58. Samosornsuk, N.; Lulitanond, A.; Saenla, N.; Anuntagool, N.; Wongratanacheewin, S.; Sirisinha, S. Short report: Evaluation of a monoclonal antibody-based latex agglutination test for rapid diagnosis of septicemic melioidosis. Am. J. Trop. Med. Hyg. 1999, 61, 735–737. [CrossRef][PubMed] 59. Kenfack, M.T.; Mazur, M.; Nualnoi, T.; Shaffer, T.L.; Ngassimou, A.; Bleriot, Y.; Marrot, J.; Marchetti, R.; Sintiprungrat, K.; Chantratita, N.; et al. Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens. Nat. Commun. 2017, 8. [CrossRef] Vaccines 2018, 6, 5 20 of 20

60. Marchetti, R.; Dillon, M.J.; Burtnick, M.N.; Hubbard, M.A.; Kenfack, M.T.; Bleriot, Y.; Gauthier, C.; Brett, P.J.; AuCoin, D.P.; Lanzetta, R.; et al. Burkholderia pseudomallei capsular polysaccharide recognition by a monoclonal antibody reveals key details toward a biodefense vaccine and diagnostics against melioidosis. ACS Chem. Biol. 2015, 10, 2295–2302. [CrossRef][PubMed] 61. Novem, V.; Shui, G.; Wang, D.; Bendt, A.K.; Sim, S.H.; Liu, Y.; Thong, T.W.; Sivalingam, S.P.; Ooi, E.E.; Wenk, M.R.; et al. Structural and biological diversity of lipopolysaccharides from Burkholderia pseudomallei and Burkholderia thailandensis. Clin. Vaccine Immunol. CVI 2009, 16, 1420–1428. [CrossRef][PubMed] 62. Weehuizen, T.A.; Prior, J.L.; van der Vaart, T.W.; Ngugi, S.A.; Nepogodiev, S.A.; Field, R.A.; Kager, L.M.; van’t Veer, C.; de Vos, A.F.; Wiersinga, W.J. Differential toll-like receptor-signalling of Burkholderia pseudomallei lipopolysaccharide in murine and human models. PLoS ONE 2015, 10, e0145397. [CrossRef][PubMed] 63. Finkelman, F.D.; Holmes, J.; Katona, I.M.; Urban, J.F., Jr.; Beckmann, M.P.; Park, L.S.; Schooley, K.A.; Coffman, R.L.; Mosmann, T.R.; Paul, W.E. Lymphokine control of in vivo immunoglobulin isotype selection. Annu. Rev. Immunol. 1990, 8, 303–333. [CrossRef][PubMed] 64. Fernandes, P.J.; Guo, Q.; Waag, D.M.; Donnenberg, M.S. The type iv pilin of Burkholderia mallei is highly immunogenic but fails to protect against lethal aerosol challenge in a murine model. Infect. Immun. 2007, 75, 3027–3032. [CrossRef][PubMed] 65. Bishop, A.L.; Schild, S.; Patimalla, B.; Klein, B.; Camilli, A. Mucosal immunization with vibrio cholerae outer membrane vesicles provides maternal protection mediated by antilipopolysaccharide antibodies that inhibit bacterial motility. Infect. Immun. 2010, 78, 4402–4420. [CrossRef][PubMed] 66. Holst, J.; Martin, D.; Arnold, R.; Huergo, C.C.; Oster, P.; O’Hallahan, J.; Rosenqvist, E. Properties and clinical performance of vaccines containing outer membrane vesicles from neisseria meningitidis. Vaccine 2009, 27, B3–B12. [CrossRef][PubMed] 67. Alving, C.R.; Rao, M. Lipid a and liposomes containing lipid a as antigens and adjuvants. Vaccine 2008, 26, 3036–3045. [CrossRef][PubMed] 68. Alving, C.R. Vaccine Adjuvants. In Vaccines for Biodefense and Emerging and Neglected Diseases; Stanberry, L.R., Ed.; Academic Press: London, UK, 2009; pp. 115–129. 69. Warawa, J.M. Evaluation of surrogate animal models of melioidosis. Front. Microbiol. 2010, 1, 141. [CrossRef] [PubMed] 70. Leakey, A.K.; Ulett, G.C.; Hirst, R.G. Balb/c and c57bl/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb. Pathog. 1998, 24, 269–275. [CrossRef][PubMed] 71. Locksley, R.M.; Heinzel, F.P.; Sadick, M.D.; Holaday, B.J.; Gardner, K.D., Jr. Murine cutaneous leishmaniasis: Susceptibility correlates with differential expansion of helper t-cell subsets. Ann. Inst. Pasteur. Immunol. 1987, 138, 744–749. [CrossRef] 72. Mills, C.D.; Kincaid, K.; Alt, J.M.; Heilman, M.J.; Hill, A.M. M-1/m-2 macrophages and the th1/th2 paradigm. J. Immunol. 2000, 164, 6166–6173. [CrossRef][PubMed] 73. Mattner, F.; Magram, J.; Ferrante, J.; Launois, P.; Di Padova, K.; Behin, R.; Gately, M.K.; Louis, J.A.; Alber, G. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized th2 cell response. Eur. J. Immunol. 1996, 26, 1553–1559. [CrossRef][PubMed] 74. Elkins, K.L.; Cooper, A.; Colombini, S.M.; Cowley, S.C.; Kieffer, T.L. In vivo clearance of an intracellular bacterium, francisella tularensis lvs, is dependent on the p40 subunit of interleukin-12 (il-12) but not on il-12 p70. Infect. Immun. 2002, 70, 1936–1948. [CrossRef][PubMed] 75. Alves, N.J.; Turner, K.B.; Medintz, I.L.; Walper, S.A. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles. Sci. Rep. 2016, 6, 24866. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).