Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria.

Novel classes of antibiotics and new strategies to prevent and treat infections are urgently needed because the rapid rise in drug-resistant bacterial infections in recent decades has been accompanied by a parallel decline in development of new antibiotics. Membrane permeabilizing antimicrobial peptides (AMPs) have long been considered a potentially promising, novel class of antibiotic, especially for wound protection and treatment to prevent the development of serious infections. Yet, despite thousands of known examples, AMPs have only infrequently proceeded as far as clinical trials, especially the chemically simple, linear examples. In part, this is due to impediments that often limit their applications in vivo. These can include low solubility, residual toxicity, susceptibility to proteolysis, and loss of activity due to host cell, tissue, and protein binding. Here we show how synthetic molecular evolution can be used to evolve potentially advantageous antimicrobial peptides that lack these impediments from parent peptides that have at least some of them. As an example of how the antibiotic discovery pipeline can be populated with more promising candidates, we evolved and optimized one family of linear AMPs into a new generation with high solubility, low cytotoxicity, potent broad-spectrum sterilizing activity against a panel of gram-positive and gram-negative ESKAPE pathogens, and antibiofilm activity against gram-positive and gram-negative biofilms. The evolved peptides have these activities in vitro even in the presence of concentrated host cells and also in vivo in the complex, cell- and protein-rich environment of a purulent animal wound model infected with drug-resistant bacteria.

The Remarkable Innate Resistance of Burkholderia bacteria to Cationic Antimicrobial Peptides: Insights into the Mechanism of AMP Resistance.

Gram-negative bacteria belonging to the genus Burkholderia are remarkably resistant to broad-spectrum, cationic, antimicrobial peptides (AMPs). It has been proposed that this innate resistance is related to changes in the outer membrane lipopolysaccharide (OM LPS), including the constitutive, essential modification of outer membrane Lipid A phosphate groups with cationic 4-amino-4-deoxy-arabinose. This modification reduces the overall negative charge on the OM LPS which may change the OM structure and reduce the binding, accumulation, and permeation of cationic AMPs. Similarly, the Gram-negative pathogen Pseudomonas aeruginosa can quickly become resistant to many AMPs by multiple mechanisms, frequently, including activation of the arn operon, which leads, transiently, to the same modification of Lipid A. We recently discovered a set of synthetically evolved AMPs that do not invoke any resistance in P. aeruginosa over multiple passages and thus are apparently not inhibited by aminorabinosylation of Lipid A in P. aeruginosa. Here we test these resistance-avoiding peptides, within a set of 18 potent AMPs, against Burkholderia thailandensis. We find that none of the AMPs tested have measurable activity against B. thailandensis. Some were inactive at concentrations as high as 150 µM, despite all having sterilizing activity at ≤ 10 µM against a panel of common, human bacterial pathogens, including P. aeruginosa. We speculate that the constitutive modification of Lipid A in members of the Burkholderia genus is only part of a broader set of modifications that change the architecture of the OM to provide such remarkable levels of resistance to cationic AMPs.

Inhibition of Streptococcus mutans biofilms with bacterial-derived outer membrane vesicles.

BACKGROUND: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. RESULTS: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced. CONCLUSION: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms.

Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4+ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen's ability to persist in vivo We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4+ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.

Consensus on the development of vaccines against naturally acquired melioidosis.

Several candidates for a vaccine against Burkholderia pseudomallei, the causal bacterium of melioidosis, have been developed, and a rational approach is now needed to select and advance candidates for testing in relevant nonhuman primate models and in human clinical trials. Development of such a vaccine was the topic of a meeting in the United Kingdom in March 2014 attended by international candidate vaccine developers, researchers, and government health officials. The focus of the meeting was advancement of vaccines for prevention of natural infection, rather than for protection from the organism's known potential for use as a biological weapon. A direct comparison of candidate vaccines in well-characterized mouse models was proposed. Knowledge gaps requiring further research were identified. Recommendations were made to accelerate the development of an effective vaccine against melioidosis.

Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

Non-mucosal vaccination strategies to enhance mucosal immunity.

The SARS-CoV-2 pandemic has highlighted the need for improved vaccines that can elicit long-lasting mucosal immunity. Although mucosal delivery of vaccines represents a plausible method to enhance mucosal immunity, recent studies utilizing intradermal vaccine delivery or incorporation of unique adjuvants suggest that mucosal immunity may be achieved by vaccination via non-mucosal routes. In this expert insight, we highlight emerging evidence from pre-clinical studies that warrant further mechanistic investigation to improve next-generation vaccines against mucosal pathogens, especially those with pandemic potential.

Mycobacterium bovis bacille Calmette-Guerin-derived extracellular vesicles as an alternative to live BCG immunotherapy.

For over 40 years, the gold standard treatment for non-muscular invasive bladder cancer (NMIBC) has been repeated administration of Mycobacterium bovis bacille Calmette-Guerin (BCG). Upon administration, BCG initiates a cascade of immunological events that lead to the recruitment of immune cells to the bladder that eliminates NMIBC cells in a multi-mechanistic, yet incompletely defined manner. Despite its effectiveness, live BCG immunotherapy is often impacted by limited supply and availability and can cause rare but serious side effects. Bacterial extracellular vesicles (EV) are nanoparticles secreted by live bacteria. EVs are composed of multiple surface proteins, sugars, and lipid that can elicit cellular responses and host recognition similar to live bacteria. In this study, we sought to evaluate the cellular responses of epithelial bladder cancer cells (BCC) to BCG EVs and live BCG. We compared the effect of each treatment on BCC cytokine production, cellular viability and apoptosis. Our data suggest that BCG EVs are as effective as live BCG in eliciting cytokine responses and halting cancer cell growth by, in part, inducing apoptosis. These results indicate that BCG EVs warrant investigation as an alternative to live BCG for NMIBC immunotherapy.

Establishment of isotype-switched, antigen-specific B cells in multiple mucosal tissues using non-mucosal immunization.

Although most pathogens infect the human body via mucosal surfaces, very few injectable vaccines can specifically target immune cells to these tissues where their effector functions would be most desirable. We have previously shown that certain adjuvants can program vaccine-specific helper T cells to migrate to the gut, even when the vaccine is delivered non-mucosally. It is not known whether this is true for antigen-specific B cell responses. Here we show that a single intradermal vaccination with the adjuvant double mutant heat-labile toxin (dmLT) induces a robust endogenous, vaccine-specific, isotype-switched B cell response. When the vaccine was intradermally boosted, we detected non-circulating vaccine-specific B cell responses in the lamina propria of the large intestines, Peyer's patches, and lungs. When compared to the TLR9 ligand adjuvant CpG, only dmLT was able to drive the establishment of isotype-switched resident B cells in these mucosal tissues, even when the dmLT-adjuvanted vaccine was administered non-mucosally. Further, we found that the transcription factor Batf3 was important for the full germinal center reaction, isotype switching, and Peyer's patch migration of these B cells. Collectively, these data indicate that specific adjuvants can promote mucosal homing and the establishment of activated, antigen-specific B cells in mucosal tissues, even when these adjuvants are delivered by a non-mucosal route. These findings could fundamentally change the way future vaccines are formulated and delivered.

Optimization of Host Cell-Compatible, Antimicrobial Peptides Effective against Biofilms and Clinical Isolates of Drug-Resistant Bacteria.

Here, we describe the continued synthetic molecular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called d-amino acid CONsensus with Glycine Absent (d-CONGA), which has excellent antimicrobial activities in vitro and in vivo. In this newest generation of rational d-CONGA variants, we tested multiple sequence-structure-function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, d-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, d-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of Klebsiella pneumoniae, innate resistance to d-CONGA was surprisingly common despite a lack of inducible resistance in Pseudomonas aeruginosa reported previously. Yet, these same isolates were susceptible to d-CONGA-Q7. d-CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, d-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile.

Microglia activation by SIV-infected macrophages: alterations in morphology and cytokine secretion.

HIV infection in the brain and the resultant encephalitis affect approximately one third of individuals infected with HIV, regardless of treatment with antiretroviral drugs. Microglia are the resident phagocytic cell type in the brain, serving as a "first responder" to neuroinvasion by pathogens. The early events of the microglial response to productively infected monocyte/macrophages entering the brain can best be investigated using in vitro techniques. We hypothesized that activation of microglia would be specific to the presence of simian immunodeficiency virus (SIV)-infected macrophages as opposed to responses to macrophages in general. Purified microglia were grown and stimulated with control or SIV-infected macrophages. After 6 h, aliquots of the supernatant were analyzed for 23 cytokines using Millipore nonhuman primate-specific kit. In parallel experiments, morphologic changes and cytokine expression by individual microglia were examined by immunofluorescence. Surprisingly, the presence of macrophages was more important to the microglial response rather than whether the macrophages were infected with SIV. None of the cytokines examined were unique to co-incubation with SIV-infected macrophages compared with control macrophages, or their supernatants. Media from SIV-infected macrophages, however, did induce secretion of higher levels of IL-6 and IL-8 than the other treatments. As resident macrophages in the brain, microglia would be expected to have a strong response to infiltrate innate immune cells such as monocyte/macrophages. This response is triggered by incubation with macrophages, irrespective of whether or not they are infected with SIV, indicating a rapid, generalized immune response when infiltrating macrophages entering the brain.

Histologic and biomechanical evaluation of biologic meshes following colonization with Pseudomonas aeruginosa.

BACKGROUND: Biologic meshes have become increasingly popular for the repair of abdominal wall defects, especially in contaminated sites. The purpose of this study was to evaluate the histologic and biomechanical properties of biologic mesh in response to a bacterial encounter. MATERIAL AND METHODS: A rat model of Pseudomonas aeruginosa colonization and infection of subcutaneously implanted biologic mesh was used. Samples of biologic meshes [acellular human dermis (ADM) and porcine small intestine submucosa (SIS)] were inoculated with P. aeruginosa (10(5) or 10(9) cfu) or saline as a control prior to wound closure (n = 6 per group). After 10 or 20 d, the meshes were harvested. The recovered meshes were analyzed for histologic changes and bacterial recovery as well as the material strength properties. Statistical significance (P < 0.05) was determined using 1-way analysis of variance or Mann-Whitney test. RESULTS: ADM and SIS colonized with 10(9) cfu P. aeruginosa showed an increased inflammatory response with an associated decrease in neo-vascularization (P < 0.05) at 20 d post-implantation compared with controls. P. aeruginosa had no effect on the tensile strength of ADM, but the tensile strength and modulus of elasticity were reduced for SIS compared with controls at 20 d. CONCLUSION: Bacterial colonization of ADM and SIS with 10(9)cfu P. aeruginosa negatively effected neovascularization and cellular re-population of the material over time but only SIS showed alterations in their biomechanical properties in response to this gram-negative bacterial challenge.

Vaccination to Prevent Pseudomonas aeruginosa Bloodstream Infections.

The bacterium Pseudomonas aeruginosa (Pa) is ubiquitous in the environment and causes opportunistic infections in humans. Pa is increasingly becoming one of the most difficult to treat microorganisms due to its intrinsic and acquired resistance to multiple antibiotics. The World Health Organization estimates that at least 700,000 people die each year from drug resistant microbial infections and have listed Pa as one of three bacterial species for which there is the most critical need for the development of novel therapeutics. Pa is a common cause of bloodstream infections (BSI) and bacterial sepsis. With nearly 49 million sepsis cases and 11 million deaths worldwide, an effective vaccine against Pa could prevent the morbidity and mortality resulting from Pa BSI and lessen our dependence on antibiotics. We reviewed the current landscape of Pa vaccines in pre-clinical and clinical stages over the last two decades. It is readily apparent that Pa vaccine development efforts have been largely directed at the prevention of pulmonary infections, likely due to Pa's devastating impact on individuals with cystic fibrosis. However, the increase in nosocomial infections, BSI-related sepsis, and the emergence of widespread antibiotic resistance have converged as a major threat to global public health. In this perspective, we draw attention to potential Pa vaccine candidates and encourage a renewed effort for prophylactic vaccine development to prevent drug-resistant Pa BSI.

The role of the Pseudomonas aeruginosa hypermutator phenotype on the shift from acute to chronic virulence during respiratory infection.

Chronic respiratory infection (CRI) with Pseudomonas aeruginosa (Pa) presents many unique challenges that complicate treatment. One notable challenge is the hypermutator phenotype which is present in up to 60% of sampled CRI patient isolates. Hypermutation can be caused by deactivating mutations in DNA mismatch repair (MMR) genes including mutS, mutL, and uvrD. In vitro and in vivo studies have demonstrated hypermutator strains to be less virulent than wild-type Pa. However, patients colonized with hypermutators display poorer lung function and a higher incidence of treatment failure. Hypermutation and MMR-deficiency create increased genetic diversity and population heterogeneity due to elevated mutation rates. MMR-deficient strains demonstrate higher rates of mucoidy, a hallmark virulence determinant of Pa during CRI in cystic fibrosis patients. The mucoid phenotype results from simple sequence repeat mutations in the mucA gene made in the absence of functional MMR. Mutations in Pa are further increased in the absence of MMR, leading to microcolony biofilm formation, further lineage diversification, and population heterogeneity which enhance bacterial persistence and host immune evasion. Hypermutation facilitates the adaptation to the lung microenvironment, enabling survival among nutritional complexity and microaerobic or anaerobic conditions. Mutations in key acute-to-chronic virulence "switch" genes, such as retS, bfmS, and ampR, are also catalyzed by hypermutation. Consequently, strong positive selection for many loss-of-function pathoadaptive mutations is seen in hypermutators and enriched in genes such as lasR. This results in the characteristic loss of Pa acute infection virulence factors, including quorum sensing, flagellar motility, and type III secretion. Further study of the role of hypermutation on Pa chronic infection is needed to better inform treatment regimens against CRI with hypermutator strains.

SARS-CoV-2 Epitopes following Infection and Vaccination Overlap Known Neutralizing Antibody Sites.

Identification of epitopes targeted following virus infection or vaccination can guide vaccine design and development of therapeutic interventions targeting functional sites, but can be laborious. Herein, we employed peptide microarrays to map linear peptide epitopes (LPEs) recognized following SARS-CoV-2 infection and vaccination. LPEs detected by nonhuman primate (NHP) and patient IgMs after SARS-CoV-2 infection extensively overlapped, localized to functionally important virus regions, and aligned with reported neutralizing antibody binding sites. Similar LPE overlap occurred after infection and vaccination, with LPE clusters specific to each stimulus, where strong and conserved LPEs mapping to sites known or likely to inhibit spike protein function. Vaccine-specific LPEs tended to map to sites known or likely to be affected by structural changes induced by the proline substitutions in the mRNA vaccine's S protein. Mapping LPEs to regions of known functional importance in this manner may accelerate vaccine evaluation and discovery of targets for site-specific therapeutic interventions.

Interleukin-10 alters effector functions of multiple genes induced by Borrelia burgdorferi in macrophages to regulate Lyme disease inflammation.

Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced by B. burgdorferi in macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and live B. burgdorferi spirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL10, MIP-1α/CCL3, granulocyte colony-stimulating factor (G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-α), IL-1α, IL-1ß, gamma interferon (IFN-γ), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced by B. burgdorferi in macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

Bacterial-Derived Outer Membrane Vesicles are Potent Adjuvants that Drive Humoral and Cellular Immune Responses.

Discovery and development of novel adjuvants that can improve existing or next generation vaccine platforms have received considerable interest in recent years. In particular, adjuvants that can elicit both humoral and cellular immune responses would be particularly advantageous because the majority of licensed vaccines are formulated with aluminum hydroxide (alum) which predominantly promotes antibodies. We previously demonstrated that bacterial-derived outer membrane vesicles (OMV) possess inherent adjuvanticity and drive antigen-specific antibody and cellular immune responses to OMV components. Here, we investigated the ability of OMVs to stimulate innate and adaptive immunity and to function as a stand-alone adjuvant. We show that OMVs are more potent than heat-inactivated and live-attenuated bacteria in driving dendritic cell activation in vitro and in vivo. Mice immunized with OMVs admixed with heterologous peptides generated peptide-specific CD4 and CD8 T cells responses. Notably, OMV adjuvant induced much greater antibody and B cell responses to co-delivered ovalbumin compared to the responses elicited by the adjuvants alum and CpG DNA. Additionally, pre-existing antibodies raised against the OMVs did not impair OMV adjuvanticity upon repeat immunization. These results indicate that vaccines adjuvanted with OMVs elicit robust cellular and humoral immune responses, supporting further development of OMV adjuvant for use in next-generation vaccines.

An Outer Membrane Vesicle-Adjuvanted Oral Vaccine Protects Against Lethal, Oral Salmonella Infection.

Non-typhoidal salmonellosis, caused by Salmonella enterica serovar Typhimurium is a common fecal-oral disease characterized by mild gastrointestinal distress resulting in diarrhea, chills, fever, abdominal cramps, head and body aches, nausea, and vomiting. Increasing incidences of antibiotic resistant invasive non-typhoidal Salmonella infections makes this a global threat requiring novel treatment strategies including next-generation vaccines. The goal of the current study was to formulate a novel vaccine platform against Salmonella infection that could be delivered orally. To accomplish this, we created a Salmonella-specific vaccine adjuvanted with Burkholderia pseudomallei outer membrane vesicles (OMVs). We show that adding OMVs to a heat-killed oral Salmonella vaccine (HKST + OMVs) protects against a lethal, oral challenge with Salmonella. Further, we show that opsonizing anti-Salmonella antibodies are induced in response to immunization and that CD4 T cells and B cells can be induced when OMVs are used as the oral adjuvant. This study represents a novel oral vaccine approach to combatting the increasing problem of invasive Salmonella infections.

Burkholderia pseudomallei OMVs derived from infection mimicking conditions elicit similar protection to a live-attenuated vaccine.

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus that causes the disease melioidosis. B. pseudomallei expresses a number of proteins that contribute to its intracellular survival in the mammalian host. We previously demonstrated that immunization with OMVs derived from B. pseudomallei grown in nutrient-rich media protects mice against lethal disease. Here, we evaluated if OMVs derived from B. pseudomallei grown under macrophage-mimicking growth conditions could be enriched with intracellular-stage proteins in order to improve the vaccine. We show that OMVs produced in this manner (M9 OMVs) contain proteins associated with intracellular survival yet are non-toxic to living cells. Immunization of mice provides significant protection against pulmonary infection similar to that achieved with a live attenuated vaccine and is associated with increased IgG, CD4+, and CD8+ T cells. OMVs possess inherent adjuvanticity and drive DC activation and maturation. These results indicate that M9 OMVs constitute a new promising vaccine against melioidosis.

Differential susceptibility of inbred mouse strains to Burkholderia thailandensis aerosol infection.

Burkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial disease that accounts for high rates of septicaemia and death in parts of Southeast Asia and Northern Australia. The closely related species Burkholderia thailandensis is considered avirulent in humans and has been used as a surrogate for B. pseudomallei in several studies. The pathogenesis of B. pseudomallei and the role of Toll-like receptors (TLRs) in host immunity to infection are not well-defined. In this study, we exposed four strains of inbred mice (BALB/c, C57BL/6, TLR4-deficient C3H/HeJ, and TLR4-competent C3H/HeN) to increasing doses of aerosolized B. thailandensis to determine strain susceptibility and the role of TLR4 during pulmonary infection. Our results indicate an increased susceptibility in the C57BL/6 and BALB/c strains, who displayed lethality, bacterial burden in organs, and pulmonary and systemic inflammation. C3H/HeJ were as resistant as C3H/HeN mice to B. thailandensis at the highest challenge dose examined, but TLR4-deficient animals exhibited a modest increase in chronic pulmonary inflammation. These results demonstrate that B. thailandensis can be used as a surrogate for experimental laboratory investigation of melioidosis in small animal models and that TLR4 may not play a prominent role during acute pneumonic melioidosis.