Keywords

1 Melioidosis

Melioidosis, also known as Whitmore’s disease, is caused by Burkholderia pseudomallei, a Gram-negative aerobic bacillus capable of infecting both animals and humans. The bacterium has an impressive collection of virulence factors encoded on two chromosomes, an unusual feature for most bacteria [1]. B. pseudomallei can cause a wide variety of disease manifestations, ranging from asymptomatic infection to life-threatening pneumonia or sepsis, which may be influenced by the route of infection and underlying human risk factors or immune status [2].

Melioidosis is most common in subtropical areas, with the highest incidence in Southeast Asia and Northern Australia. B. pseudomallei is also found in parts of Africa and has recently been detected in the US Virgin Islands and other Caribbean islands, including Puerto Rico [3]. While B. pseudomallei is not considered endemic in Europe or North America, four cases of melioidosis were recently reported in the USA. The four cases occurred in Kansas, Texas, Georgia, and Minnesota in individuals who had not traveled outside of the USA. Genetic analyses of the isolates linked the infection to a common source—a contaminated imported aromatherapy spray [4, 5]. There have been case reports of endemic melioidosis in Central and South America, pointing to a more global spread of B. pseudomallei than previously appreciated [6].

B. pseudomallei is a soil saprophyte, and routes of exposure may include inoculation, inhalation, or ingestion from a contaminated source. The incubation time to disease onset is 1–21 days from exposure with an average of 9 days, depending on the infectious dose and host factors. Melioidosis is considered an acute infection if the symptoms manifest for less than 2 months, whereas symptoms persisting longer than 2 months constitute a chronic infection. Latent infection with reactivation disease can also occur [7]. The outcomes of infection depend on several factors including B. pseudomallei strain, route of infection and dose, and host immune status. The most common host risk factor in development of severe melioidosis is diabetes mellitus [8], but other risk factors include chronic kidney, lung, and liver diseases and other conditions that impair the immune system [9]. Nonetheless, severe sickness and death can also occur in otherwise healthy individuals [10]. The case fatality rate percentage (CFR%) ranges from 8% in Europe up to 60% in Africa. In endemic areas, such as Australia, the CFR% is approximately 41% [11].

Melioidosis may result in a broad range of symptoms, such as pneumonia, fever, pain, cough, and headache. The disease often mimics the manifestations of more common pathogens leading to misdiagnosis and delayed treatment [2, 12]. Diagnosis based only on clinical manifestations is sometimes possible, but bacterial culture is the gold standard for proper diagnosis. Because of the nature of Burkholderia species, it may sometimes be dismissed as a contaminant, so appropriate training of clinical laboratory staff in endemic areas is important. In a presumed melioidosis case, clinical samples should be sent to laboratories with experience in identifying the bacterium. Common samples include blood, throat or wound swabs, and urine. Repeated sampling is needed as samples may initially test negative. B. pseudomallei grows slowly on most agar plates and may take a few days for visible detection. Antibiotic resistance profiles are helpful for identification, especially in settings with scarce resources. B. pseudomallei is resistant to gentamicin and polymyxin and sensitive to amoxicillin-clavulanic acid. This sensitivity pattern in a Gram-negative, oxidase-positive bacillus can be helpful for diagnosis. Molecular diagnostics are not common in most endemic areas. Proper clinical management must be initiated early in every suspect case. B. pseudomallei is susceptible to β-lactam antibiotics such as imipenem, meropenem, amoxicillin-clavulanic acid, and ceftazidime. In the eradication phase, trimethoprim/sulfamethoxazole is recommended [13].

Prevention may be achieved in endemic areas by avoiding high-risk activities and through additional protective measures for those at occupational risk. Often, this is difficult to achieve in poor countries with high B. pseudomallei endemicity. There is currently no licensed vaccine to prevent melioidosis. A vaccine could mitigate the public health burden of B. pseudomallei in areas of the world with high caseloads. In addition, B. pseudomallei has been classified as a Tier 1 select agent by the US Department of Health and Human Services and the US Department of Agriculture, due to its potential threat as a bioweapon. A vaccine to safeguard against B. pseudomallei misuse is therefore also highly desired.

2 Pathogenesis and Host Response

B. pseudomallei utilizes multiple virulence factors to invade human cells. This bacterium can enter mammalian cells via adherence using adhesion proteins such as fimbriae, type IV pili, flagellin [14,15,16], and components of the type V secretion system that work as autotransporters, such as BcaA, and the trimeric autotransporter adhesins BoaA and BoaB [17]. The type III secretion system proteins are used to invade and escape the endosome and are required for intracellular survival [18]. Type II, type III, and type VI secretion systems are required for the bacteria to exit the cells by membrane lysis [19, 20]. Autophagy is avoided by the effector protein BopA and translocator protein BipD [21,22,23]. Actin-based motility is mediated by BimA and leads to cell-to-cell fusion and the formation of multinucleated giant cells that are associated with granulomatous diseases, such as sarcoidosis and tuberculosis, complicating clinical diagnosis [24].

Immune responses to B. pseudomallei infection are triggered by pathogen-associated molecular patterns (PAMP) activation upon recognition of bacterial lipoproteins, lipopolysaccharide (LPS), and flagella by Toll-like receptor (TLR)2, TLR4, and TLR5, respectively, leading to a pro-inflammatory response mediated via NF-κΒ activation [25, 26]. Activation of complement pathway and destruction via the membrane attack complex is impaired by the presence of capsular polysaccharide (CPS), a major virulence determinant [27]. Activation of caspase 1 via the inflammasome leads to pyroptosis, releasing IL-1β and IL-18 [28, 29]. IL-18 promotes the induction of IFN-γ, helping to recruit additional macrophages. Macrophages that are activated via INF-γ produce nitric oxide that can kill sensitive strains [30]. However, some strains have developed resistance to oxidative stress by expressing superoxide dismutase C (sodC), alkyl hydroperoxide reductase C (ahpC), and a nonspecific DNA-binding protein known as dpsA [31, 32]. The release of IL-1β contributes to pathogenesis by recruiting neutrophils, which leads to tissue damage [33]. As commonly observed in other diseases, uncontrolled pro-inflammatory responses are associated with poor outcomes. An exacerbated release of IL-1β, IL-6, IL-12, TNF, and IFN-γ can cause severe melioidosis disease [27]. Both humoral and cell-mediated immune responses have been shown to protect against disease. Antibodies against LPS, O-polysaccharide (OPS), and Hcp-1 of B. pseudomallei are associated with better outcomes in melioidosis [27, 34]. CD4+ T cells that secrete IFN-γ are found in acute melioidosis patients and are thought to play a role in protection [35, 36]. However, further research is required to elucidate the innate or adaptive immune responses that are responsible for full protection against B. pseudomallei.

3 Vaccine Development

Over the last decade, several promising vaccine candidates have emerged using various approaches, and the most efficacious vaccines appear to be multivalent in nature. This is not entirely surprising, considering the sophisticated intracellular pathogenesis of B. pseudomallei that may require both arms of the immune response to target multiple antigens for complete protection. The various vaccine platforms include, but are not limited to, live-attenuated or inactivated whole-cell bacteria and multivalent subunit or conjugate vaccines. These are summarized below and listed in Table 15.1. The target bacterial antigens and their respective functions are described in Table 15.2.

Table 15.1 Vaccine candidates developed and tested for Burkholderia pseudomallei. The vaccine studies described here are not prioritized in any way
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Table 15.2 Antigens used in the vaccines covered in this chapter and their function
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3.1 Live-Attenuated Vaccines

A successful vaccine against B. pseudomallei will likely require both humoral and cellular immune responses for complete protection. Live-attenuated vaccines have long been considered the gold standard for achieving both arms of the immune response. Live-attenuated vaccines created by mutation of tonB and hcp1, genes belonging to a siderophore complex and the T6SS, respectively, induced robust immunity and significant protection in multiple studies [57, 58]. Deletion of purM created a highly attenuated strain (Bp82) that was avirulent in mice, Syrian hamsters, and immune incompetent mice [interferon (IFN)-γ−/−, severe combined immunodeficiency (SCID)] [59]. Immunization of mice with Bp82 conferred 100% survival over 60 days against pulmonary melioidosis [60]. Deletion of relA-spoT produced a B. pseudomallei double mutant that displayed defects in stationary-phase survival and replication in murine macrophages and was attenuated in acute and chronic mouse models of melioidosis. Immunization of mice with the ΔrelA ΔspoT live-attenuated strain resulted in full protection against infection with B. pseudomallei [39]. An asd mutant of B. pseudomallei 1026b was avirulent in BALB/c mice, and animals vaccinated with the mutant were protected against acute inhalation melioidosis [40]. Immunization with an aroC mutant also significantly protected mice against challenge [38]. Immunization with B. thailandensis strain E555, which possesses CPS, provided significant protection against B. pseudomallei challenge [42]. Collectively, these studies demonstrate the safety and protective efficacy of live-attenuated vaccines in animal models of melioidosis.

3.2 Inactivated Whole-Cell Vaccines

Historically, whole-cell, inactivated vaccines have not provided the same level of protection as live vaccines in rodent models of melioidosis. This has prompted novel approaches to improve their protective efficacy. Incorporation of cationic liposomes complexed with noncoding plasmid DNA (CLDC) to a heat-killed B. pseudomallei vaccine conferred 100% survival of immunized mice that lasted greater than 40 days [37]. In another study, synthetic microparticles composed of acetylated dextran with encapsulated B. pseudomallei cell lysate induced significant protection against a lethal challenge in mice [56].

3.3 Subunit Vaccines

One of the vaccine targets that have been extensively studied is the CPS, a known protective antigen and virulence determinant for B. pseudomallei. The CPS is a homopolymer of unbranched 1 → 3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Immunization with B. pseudomallei CPS linked to CRM197, a nontoxic mutant of diphtheria toxin, elicited IgG and opsonizing antibody responses. Immunization with a combination of CPS-CRM197 and recombinant Hcp1 protected 100% of mice following an otherwise lethal inhalation challenge with B. pseudomallei [46]. In another study, immunization with chemically synthesized CPS conjugated to tetanus toxoid induced serum IgM and IgG antibodies. While the CPS-specific antibody titers were considerably less than that induced by native CPS, 66% of BALB/c mice survived challenge with B. pseudomallei [48].

Immunization with lipopolysaccharide (LPS) conjugated to the Hc fragment of tetanus toxoid produced a better survival outcome (81%) compared to immunization with LPS alone (62%) [44]. B. pseudomallei O-polysaccharide (OPS II) conjugated to a carrier protein AcrA from Campylobacter jejuni elicited similar levels of protection to killed bacteria against a highly lethal challenge [49]. LPS glycoconjugates have also been successfully incorporated onto the surface of gold nanoparticles (AuNP). Mice immunized with AuNP-FlgL-LPS alone or with a protein combination (FlgL, Hcp1, and hemagglutinin) demonstrated up to 100% survival and reduced lung colonization following a lethal challenge with B. pseudomallei [51].

Other protein subunits have been identified as possible candidates due to their immunogenicity, such as FliC, BopE, AhpC, and several outer membrane proteins (OMP) [62,63,64,65,66,66]. A DNA vaccine composed of flagellin reduced bacterial burdens, lung inflammation, and pathology. Levels of plasma TNF-α, IFN-γ, and MCP1 cytokines were also reduced, signaling a reduction in systemic inflammation. Vaccinated mice displayed 53% survival compared to 15% survival in control mice [45]. Mice immunized with a single dose of parainfluenza virus 5 (PIV5) expressing BatA, a Burkholderia autotransporter protein, displayed 60% overall survival, with 78% of surviving mice clearing bacteria in the lungs and 44% clearing bacteria in the spleen [67]. A synthetic biology approach was used to predict the antigenicity and toxicity of a recombinant collagen-like 8 protein that is conserved among Burkholderia species [68]. Two formulations were created, one with a recombinant protein and another one using the β-barrel portion of the protein. Use of β-barrel peptides from Buc (Bucl8) adjuvanted with an oil-in-water nano-emulsion produced a strong Th2 response in C57BL/6 mice.

B. pseudomallei outer membrane vesicles (OMVs) contain numerous protective antigens associated with the bacterial outer membrane including OmpA, CPS, and LPS. B. pseudomallei OMV vaccines have demonstrated safety, immunogenicity, and protection against pneumonic and septicemic disease in mice and nonhuman primates [53, 54, 69]. When OMVs are produced under macrophage mimicking growth conditions, they contain proteins that are associated with intracellular survival, conferring strong protection against inhalational melioidosis and eliciting both humoral and cellular immune responses [55]. Due to the sophisticated intracellular pathogenesis of B. pseudomallei, inclusion of antigens expressed during the chronic stage of infection may be important for obtaining complete vaccine protection. Three proteins, BPSL1897, BPSL2287, and BPSL3369, which were expressed in chronically infected mice, were combined with the surface protein OmpA (BPSL2765) to create a multicomponent subunit vaccine formulation. Mice immunized with the chronic antigens plus LolC or CPS displayed increased survival compared to mice immunized with only the individual subunits, LolC, or CPS [43].

4 Conclusions

In recent years, several promising vaccine candidates have emerged to combat melioidosis, which is considered an emerging and expanding infectious disease. Adjuvants are also being evaluated as part of vaccine development as they can drive different protective components of humoral and cellular immune responses. Going forward, it will be important to elucidate the immune mechanisms of protection to better inform vaccine design and to establish immune correlates of vaccine protection. Based on recent success in animal models, human clinical trials are currently being planned for more than one vaccine candidate. These include a phase 1 clinical trial of vaccine candidate CPS-CRM197/Hcp1 adjuvanted with alum/CpG that was developed at the University of Nevada, Reno, USA [46]. The trial will take place in Oxford in 36 healthy human subjects with a follow-up phase 1b planned in Ubon Ratchathani, Thailand. An OMV vaccine, developed at Tulane University, Louisiana, USA, is also being considered for phase 1 clinical trials planned in Australia [55]. For any vaccine, larger phase 2 and 3 clinical trials would be necessary to demonstrate vaccine safety and efficacy. Nonetheless, initiation of human vaccine trials for a disease once largely ignored sparks hope that licensure of human vaccines to prevent melioidosis may soon be realized.