LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice.

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines. IMPORTANCE: Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

Burkholderia pseudomallei and melioidosis.

Burkholderia pseudomallei, the causative agent of melioidosis, is found in soil and water of tropical and subtropical regions globally. Modelled estimates of the global burden predict that melioidosis remains vastly under-reported, and a call has been made for it to be recognized as a neglected tropical disease by the World Health Organization. Severe weather events and environmental disturbance are associated with increased case numbers, and it is anticipated that, in some regions, cases will increase in association with climate change. Genomic epidemiological investigations have confirmed B. pseudomallei endemicity in newly recognized regions, including the southern United States. Melioidosis follows environmental exposure to B. pseudomallei and is associated with comorbidities that affect the immune response, such as diabetes, and with socioeconomic disadvantage. Several vaccine candidates are ready for phase I clinical trials. In this Review, we explore the global burden, epidemiology and pathophysiology of B. pseudomallei as well as current diagnostics, treatment recommendations and preventive measures, highlighting research needs and priorities.

Development of Melioidosis Subunit Vaccines Using an Enzymatically Inactive Burkholderia pseudomallei AhpC.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular, Gram-negative pathogen that is highly infectious via the respiratory route and can cause severe, debilitating, and often fatal diseases in humans and animals. At present, no licensed vaccines for immunization against this CDC Tier 1 select agent exist. Studies in our lab have previously demonstrated that subunit vaccine formulations consisting of a B. pseudomallei capsular polysaccharide (CPS)-based glycoconjugate (CPS-CRM197) combined with hemolysin-coregulated protein (Hcp1) provided C57BL/6 mice with high-level protection against an acute inhalational challenge of B. pseudomallei. In this study, we evaluated the immunogenicity and protective capacity of B. pseudomallei alkyl hydroperoxide reductase subunit C (AhpC) in combination with CPS-CRM197. AhpC is a peroxiredoxin involved in oxidative stress reduction and is a potential protective antigen. To facilitate our studies and maximize safety in animals, recombinant B. pseudomallei AhpC harboring an active site mutation (AhpCC57G) was expressed in Escherichia coli and purified using tandem nickel-cobalt affinity chromatography. Immunization of C57BL/6 mice with CPS-CRM197 combined with AhpCC57G stimulated high-titer IgG responses against the CPS component of the glycoconjugate as well as stimulated high-titer IgG and robust interferon gamma (IFN-γ)-, interleukin-5 (IL-5)-, and IL-17-secreting T cell responses against AhpCC57G. When challenged via an inhalational route with a high dose (~27 50% lethal doses [LD50s]) of B. pseudomallei, 70% of the immunized mice survived 35 days postchallenge. Collectively, our findings demonstrate that AhpCC57G is a potent activator of cellular and humoral immune responses and may be a promising candidate to include in future melioidosis subunit vaccines.

Melioidosis in Africa: Time to Raise Awareness and Build Capacity for Its Detection, Diagnosis, and Treatment.

Melioidosis is a tropical infectious disease caused by the soil-dwelling bacterium Burkholderia pseudomallei with a mortality of up to 50% in low resource settings. Only a few cases have been reported from African countries. However, studies on the global burden of melioidosis showed that Africa holds a significant unrecognized disease burden, with Nigeria being at the top of the list. The first World Health Organization African Melioidosis Workshop was organized in Lagos, Nigeria, with representatives of health authorities, microbiology laboratories, and clinical centers from across the continent. Dedicated hands-on training was given on laboratory diagnostics of B. pseudomallei. This report summarises the meeting objectives, including raising awareness of melioidosis and building capacity for the detection, diagnosis, biosafety, treatment, and prevention across Africa. Further, collaboration with regional and international experts provided a platform for sharing ideas on best practices.

Hcp1-loaded staphylococcal membrane vesicle vaccine protects against acute melioidosis.

Burkholderia pseudomallei is the causal agent of melioidosis, a deadly tropical infectious disease that lacks a vaccine. On the basis of the attenuated Staphylococcus aureus RN4220-Δagr (RN), we engineered the RN4220-Δagr/pdhB-hcp1 strain (RN-Hcp1) to generate B. pseudomallei hemolysin-coregulated protein 1 (Hcp1)-loaded membrane vesicles (hcp1MVs). The immunization of BALB/c mice with hcp1MVs mixed with adjuvant by a three-dose regimen increased the serum specific IgG production. The serum levels of inflammatory factors, including TNF-α and IL-6, in hcp1MV-vaccinated mice were comparable with those in PBS-challenged mice. The partial adjuvant effect of staphylococcal MVs was observed with the elevation of specific antibody titer in hcp1MV-vaccinated mice relative to those that received the recombinant Hcp1 protein (rHcp1) or MVs derived from RN strain (ΔagrMVs). The hcp1MVs/adjuvant vaccine protected 70% of mice from lethal B. pseudomallei challenge. Immunization with hcp1MVs only protected 60% of mice, whereas vaccination with rHcp1 or ΔagrMVs conferred no protection. Moreover, mice that received hcp1MVs/adjuvant and hcp1MVs immunization had low serum TNF-α and IL-6 levels and no inflammatory infiltration in comparison with other groups. In addition, all surviving mice in hcp1MVs/adjuvant and hcp1MVs groups exhibited no culturable bacteria in their lungs, livers, and spleens five days postinfection. Overall, our data highlighted a new strategy for developing B. pseudomallei vaccine and showed that Hcp1-incorporated staphylococcal MV is a promising candidate for the prevention of acute melioidosis.

BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis.

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

Multicomponent Gold-Linked Glycoconjugate Vaccine Elicits Antigen-Specific Humoral and Mixed TH1-TH17 Immunity, Correlated with Increased Protection against Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal disease with a high mortality rate. The intrinsic resistance to commonly used antibiotics combined with the complex bacterial life cycle has hampered the development of preventive and therapeutic interventions and vaccines. Furthermore, the need of humoral and cell-mediated immunity in protection against B. pseudomallei has complicated the development of effective vaccines. Antigen delivery vaccine platforms that promote humoral and cellular responses while maintaining a safe profile are a roadblock to developing subunit vaccines against intracellular pathogens. Gold nanoparticles (AuNPs) were used for the delivery of multicomponent antigens with the goal of inducing vaccine-mediated immunity, promoting protection against melioidosis disease. Different nanoglycoconjugates using predicted immunogenic protein candidates, Hcp1, FlgL, OpcP, OpcP1, OmpW, and hemagglutinin, were covalently coupled to AuNPs, together with the lipopolysaccharide (LPS) from Burkholderia thailandensis, which acted as an additional antigen. Animals immunized with individually coupled (AuNP-protein-LPS) formulations containing OpcP or OpcP1, together with CpG as an adjuvant, showed a significant increase in protection, whereas a nanovaccine combination (AuNP-Combo2-LPS) showed significant and complete protection against a lethal intranasal B. pseudomallei challenge. Animals immunized with AuNP-Combo2-LPS showed robust humoral antigen-specific (IgG and IgA) responses with higher IgG2c titer, indicating a TH1-skewed response and promotion of macrophage uptake. In addition, immunization with the nanovaccine combination resulted in a mixed antigen-specific TH1-TH17 cytokine profile after immunization. This study provides the basis for an elegant and refined multicomponent glycoconjugate vaccine formulation capable of eliciting both humoral and cell-mediated responses against lethal B. pseudomallei challenge. IMPORTANCE Melioidosis is a complex human disease associated with a wide range of complications caused by the Gram-negative bacillus Burkholderia pseudomallei. The global burden of melioidosis is estimated to have 165,000 cases per year and 89,000 fatal outcomes. The endemicity of B. pseudomallei includes a wide range of tropical regions in Asia, Africa, Latin America, and Australia. Therefore, a viable alternative to prevent human infections is the development of an effective vaccine; however, no approved vaccine for human use is available. This study provides a vaccine strategy against B. pseudomallei and an immune-stimulatory platform to induce strong humoral and T-cell-mediated immunity.

Effectiveness of a multifaceted prevention programme for melioidosis in diabetics (PREMEL): A stepped-wedge cluster-randomised controlled trial.

BACKGROUND: Melioidosis, an often-fatal infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, is endemic in tropical countries. Diabetes mellitus and environmental exposure are important risk factors for melioidosis acquisition. We aim to evaluate the effectiveness of a multifaceted prevention programme for melioidosis in diabetics in northeast Thailand. METHODOLOGY/PRINCIPAL FINDINGS: From April 2014 to December 2018, we conducted a stepped-wedge cluster-randomized controlled behaviour change trial in 116 primary care units (PCUs) in Ubon Ratchathani province, northeast Thailand. The intervention was a behavioural support group session to help diabetic patients adopt recommended behaviours, including wearing rubber boots and drinking boiled water. We randomly allocated the PCUs to receive the intervention starting in March 2016, 2017 and 2018. All diabetic patients were contacted by phone yearly, and the final follow-up was December 2018. Two primary outcomes were hospital admissions involving infectious diseases and culture-confirmed melioidosis. Of 9,056 diabetics enrolled, 6,544 (72%) received a behavioural support group session. During 38,457 person-years of follow-up, we observed 2,195 (24%) patients having 3,335 hospital admissions involved infectious diseases, 80 (0.8%) melioidosis, and 485 (5%) deaths. In the intention-to-treat analysis, implementation of the intervention was not associated with primary outcomes. In the per-protocol analysis, patients who received a behavioural support group session had lower incidence rates of hospital admissions involving infectious diseases (incidence rate ratio [IRR] 0.89; 95%CI 0.80-0.99, p = 0.03) and of all-cause mortality (IRR 0.54; 95%CI 0.43-0.68, p<0.001). However, the incidence rate of culture-confirmed melioidosis was not significantly lower (IRR 0.96, 95%CI 0.46-1.99, p = 0.66). CONCLUSIONS/SIGNIFICANCE: Clear benefits of this multifaceted prevention programme for melioidosis were not observed. More compelling invitations for the intervention, modification of or addition to the behaviour change techniques used, and more frequent intervention may be needed. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov, number NCT02089152.

Laboratory Safety: Handling Burkholderia pseudomallei Isolates without a Biosafety Cabinet.

Burkholderia pseudomallei is a tier 1 select agent that is associated with laboratory-acquired melioidosis, with international guidelines recommending isolate handling within a class II biosafety cabinet (BSC) in a biosafety level 3 (BSL3) facility. In low-resource settings, this may not be practical; therefore, we aimed to assess the risk of laboratory-acquired melioidosis during routine work. Prior exposure to the organism was determined with a questionnaire and concomitant serology. Of 30 laboratory scientists handling B. pseudomallei on 1,267 occasions outside a biosafety cabinet, no infections were documented and all participants remained seronegative. Additionally, we performed controlled environmental air sampling during 78 laboratory handling events, including plate opening, oxidase testing, and McFarland suspension creation. None of the experiments demonstrated aerosolization of the organism. This study suggests the risk of laboratory-acquired melioidosis is low. However, individual laboratories will need to undertake a risk assessment, including melioidosis endemicity, availability of resources for containment, the nature of routine handling to be undertaken, and the presence of predisposing risk factors for infection in the staff concerned. Additionally, laboratories should take region-specific guidelines into consideration. Further research is required to better inform on the overall risk of infection in the microbiology laboratory.

A macrophage-targeted platform for extending drug dosing with polymer prodrugs for pulmonary infection prophylaxis.

Pulmonary melioidosis is a bacterial disease with high morbidity and a mortality rate that can be as high as 40% in resource-poor regions of South Asia. This disease burden is linked to the pathogen's intrinsic antibiotic resistance and protected intracellular localization in alveolar macrophages. Current treatment regimens require several antibiotics with multi-month oral and intravenous administrations that are difficult to implement in under-resourced settings. Herein, we report that a macrophage-targeted polyciprofloxacin prodrug acts as a surprisingly effective pre-exposure prophylactic in highly lethal murine models of aerosolized human pulmonary melioidosis. A single dose of the polymeric prodrug maintained high lung drug levels and targeted an intracellular depot of ciprofloxacin to the alveolar macrophage compartment that was sustained over a period of 7 days above minimal inhibitory concentrations. This intracellular pharmacokinetic profile provided complete pre-exposure protection in a BSL-3 model with an aerosolized clinical isolate of Burkholderia pseudomallei from Thailand. This total protection was achieved despite the bacteria's relative resistance to ciprofloxacin and where an equivalent dose of pulmonary-administered ciprofloxacin was ineffective. For the first time, we demonstrate that targeting the intracellular macrophage compartment with extended antibiotic dosing can achieve pre-exposure prophylaxis in a model of pulmonary melioidosis. This fully synthetic and modular therapeutic platform could be an important therapeutic approach with new or re-purposed antibiotics for melioidosis prevention and treatment, especially as portable inhalation devices in high-risk, resource-poor settings.

The Peptidoglycan-associated lipoprotein Pal contributes to the virulence of Burkholderia mallei and provides protection against lethal aerosol challenge.

BURKHOLDERIA MALLEI: is a highly pathogenic bacterium that causes the fatal zoonosis glanders. The organism specifies multiple membrane proteins, which represent prime targets for the development of countermeasures given their location at the host-pathogen interface. We investigated one of these proteins, Pal, and discovered that it is involved in the ability of B. mallei to resist complement-mediated killing and replicate inside host cells in vitro, is expressed in vivo and induces antibodies during the course of infection, and contributes to virulence in a mouse model of aerosol infection. A mutant in the pal gene of the B. mallei wild-type strain ATCC 23344 was found to be especially attenuated, as BALB/c mice challenged with the equivalent of 5,350 LD50 completely cleared infection. Based on these findings, we tested the hypothesis that a vaccine containing the Pal protein elicits protective immunity against aerosol challenge. To achieve this, the pal gene was cloned in the vaccine vector Parainfluenza Virus 5 (PIV5) and mice immunized with the virus were infected with a lethal dose of B. mallei. These experiments revealed that a single dose of PIV5 expressing Pal provided 80% survival over a period of 40 days post-challenge. In contrast, only 10% of mice vaccinated with a PIV5 control virus construct survived infection. Taken together, our data establish that the Peptidoglycan-associated lipoprotein Pal is a critical virulence determinant of B. mallei and effective target for developing a glanders vaccine.

Combating the great mimicker: latest progress in the development of Burkholderia pseudomallei vaccines.

Introduction Burkholderia pseudomallei is an environmental intracellular Gram-negative bacterium that causes melioidosis, a severe infectious disease affecting humans and animals. An increase in melioidosis cases worldwide and the high mortality rate of the disease makes it a public health concern. Melioidosis is known as the 'great mimicker' because it presents with a wide range of disease manifestations. B. pseudomallei is naturally resistant to antibiotics and delay in diagnosis leads to ineffective treatment. Furthermore, there is no approved vaccine to prevent melioidosis infection in humans. Therefore, it is a priority to license a vaccine that can be used for both high-risk endemic areas and for biodefense purposes. Areas covered In this review, we have focussed on recent progress in the USA for the development and advancement of lead B. pseudomallei vaccine candidate(s) ready for testing in pre-clinical trials. Those candidates include live-attenuated vaccines, glycoconjugate vaccines, outer-membrane vesicles, and gold nanoparticle vaccines. Expert opinion Side-by-side comparison of the leading B. pseudomallei vaccine candidates will provide important information to further advance studies into pre-clinical trials. The likelihood of any of these current vaccines becoming the selected candidate that will reduce the occurrence of melioidosis worldwide is closer than ever.

Melioidosis: A Neglected Cause of Community-Acquired Pneumonia.

Melioidosis, caused by the facultative intracellular gram-negative pathogen Burkholderia pseudomallei, is an emerging cause of community-acquired pneumonia across the tropics. The majority of patients present with pneumonia with or without sepsis, but localized and asymptomatic infection is also well recognized. Recent modeling and epidemiological studies have demonstrated the widespread presence of B. pseudomallei in otherwise unrecognized regions with a predicted mortality of 90,000 deaths worldwide. Innovative environmental studies are also uncovering how hydrodynamic, pedology, fauna, and weather events influence geographic distribution and incidence of melioidosis cases. Of concern is the changes associated with global warming, which will be conducive to B. pseudomallei in combination with the global diabetes pandemic. In fact, over 80% of patient developing melioidosis have underlying comorbidities. For this great mimicker, culture remains the mainstay of diagnosis and despite availability of other assays, challenges still remain in reducing time to diagnosis and avoiding misdiagnosis. With institution of timely antimicrobials such as ceftazidime and supportive intensive care, overall mortality can be reduced to 10%, although this can still be as high as 50% in poorly resourced areas. Promise is on the horizon with the first human vaccine trials being planned for 2021. Meanwhile new multiomics techniques are giving us a better understanding of the role of virulence and host-pathogen interactions on patient outcomes.

Burkholderia pseudomallei pathogenesis and survival in different niches.

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease of the tropics with high clinical mortality rates. To date, no vaccines are approved for melioidosis and current treatment relies on antibiotics. Conversely, common misdiagnosis and high pathogenicity of Bp hamper efforts to fight melioidosis. This bacterium can be isolated from a wide range of niches such as waterlogged fields, stagnant water bodies, salt water bodies and from human and animal clinical specimens. Although extensive studies have been undertaken to elucidate pathogenesis mechanisms of Bp, little is known about how a harmless soil bacterium adapts to different environmental conditions, in particular, the shift to a human host to become a highly virulent pathogen. The bacterium has a large genome encoding an armory of factors that assist the pathogen in surviving under stressful conditions and assuming its role as a deadly intracellular pathogen. This review presents an overview of what is currently known about how the pathogen adapts to different environments. With in-depth understanding of Bp adaptation and survival, more effective therapies for melioidosis can be developed by targeting related genes or proteins that play a major role in the bacteria's survival.

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

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

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

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