Vaccines for the Prevention of Melioidosis and Glanders

Abstract

Purpose of review

Burkholderia pseudomallei’s and Burkholderia mallei’s high rate of infectivity, limited treatment options, and potential use as biological warfare agents underscore the need for development of effective vaccines against these bacteria. Research efforts focused on vaccines against these bacteria are in pre-clinical stages, with no approved formulations currently on the market.

Recent findings

Several live attenuated and subunit vaccine formulations have been evaluated in animal studies, with no reports of significant long term survival after lethal challenge.

Summary

This review encompasses the most current vaccine strategies to prevent B. pseudomallei and B. mallei infections while providing insight for successful vaccines moving forward.

Introduction

Melioidosis is an infectious disease caused by Burkholderia pseudomallei and which is endemic in many tropical regions of the world [1]. B. pseudomallei, and its relative Burkholderia mallei, the causative agent of glanders, are aerobic, gram-negative, motile bacilli classified by the US Centers for Disease Control and Prevention (CDC) as Tier 1 select agents due to high virulence when inhaled, and potential to cause large-scale illness if disseminated. Additionally, Burkholderia spp. have resistance to a wide range of antibiotics, including penicillin [2, 3], which adds additional concern surrounding the bacteria. Research efforts over the last decade have evaluated prophylactic and therapeutic treatment options to combat these emerging infectious diseases; however, to date, there are no licensed vaccines on the market. This is likely due to the difficulty in designing safe and effective vaccines that protect against both acute and chronic infections caused by these pathogens. In addition, B. pseudomallei and B. mallei are biosafety level three (BSL3) agents that require additional laboratory safety precautions, further hampering vaccine development. This review will outline recent vaccine strategies to prevent infections by B. pseudomallei, and B. mallei, highlighting potential candidate vaccines that demonstrated significant protection in a model for melioidosis and glanders.

Diseases caused by Burkholderia

Members of the genus Burkholderia are ecologically diverse due to their large and dynamic genomes, allowing them to survive and persist in the environment and host cells alike [4]. B. pseudomallei is an obligate aerobe found at high concentrations in tropical regions with favorable moisture and heat conditions allowing for sustained growth [5]. Whereas B. pseudomallei is a soil-dwelling bacterium, B. mallei is an obligate animal pathogen that is non-motile [6]. Despite the differences in environmental location, they are both facultative intracellular bacteria that can survive and replicate in many cell types in the host. We next discuss how infection from these bacteria occur and highlight the challenges that must be overcome to formulate vaccines against these infectious agents.

Emergence of melioidosis is endemic in regions such as southeast Asia and northern Australia, and once patients are infected, the disease is difficult to treat do to its unique ability to infect organs and persist [7, 8]. Manifestation of symptoms depends greatly on the route of infection, with the most severe occurring due to the inhalational form of infection [9]. Acute infection with B. pseudomallei can lead to symptoms of severe pneumonia and rapid septicemia. Chronic infection can lie dormant for many years and upon re-activation, lead to fatal abscesses in multiple organs [10]. The difficulty in treating melioidosis is in part because B. pseudomallei is able to infect, replicate, and survive in a multitude of host cells, include monocytes [11, 12]. Once intracellular, the bacterium can evade endocytic vesicle transport and multiply in the cytoplasm. This advanced style of motility enables B. pseudomallei to move to different host cells and cause the formation of multi-nucleate giant cells (MNGC), resulting in toxicity and local necrosis [13]. As the infection moves to different organs such as the liver, spleen, and brain, the potential for a septic infection increases, as does the mortality due to infection [14–16].

Whereas melioidosis infection can occur via contact with infected soil or water, glanders is a zoonotic disease caused by B. mallei that requires contact with infected animals [17, 18]. However, like B. pseudomallei, B. mallei is concerning as a biothreat due to its ability to infect via inhalation resulting in high fatality. In fact, B. mallei was one of the first bioweapons utilized in the World Wars and is classified, along with B. pseudomallei, as a Tier 1 Select Agent by the CDC [19]. Although no cases of human glanders has been reported in the U.S. since the 1950’s, the fatality rate of infection were reportedly high (40%), even when properly diagnosed and treated [1, 20].

Infections by B. pseudomallei and B. mallei are inherently difficult to treat due to their resistance to broad-spectrum antibiotics, mostly as a result of efflux pump mechanisms [21]. Additionally, Burkholderia are opportunistic pathogens that can lie dormant for many years, making the establishment of intracellular chronic infections hard to treat [11]. There is a crucial need for preventative measures, such as vaccines, to provide protection to individuals living in or visiting endemic areas. Although a number of vaccine candidates have been suggested, many fail to provide complete and sterilizing immunity against both acute and chronic infections. Summarized next are strategies utilizing various antigen and adjuvant combinations to confer protection against Burkholderia spp. infections.

Live-attenuated vaccines

Live attenuated vaccines (LAV) are known for their ability to elicit potent humoral and cell-mediated immune responses that can provide long lasting protection [22, 23]. Sequencing of the Burkholderia genome has allowed researchers to genetically alter genes identified as virulence factors and exploit the resultant mutated genes as promising candidates for LAVs. These mutations are largely cellular metabolism and transport, secretion system (SS) or virulence-associated factors.

The largest portion of research on LAV mutants is altering cellular metabolism and transport. Attenuated Burkholderia mutant strains lacking functional genes in amino acid synthesis [ΔpurN [24], ΔpurM [25, 24], ΔilvI [26–28], ΔaroC [29], Δasd [30]], prokaryotic signaling [(ΔrelA/spT [31]], iron transport/intracellular survival (ΔtonB/hcp1 [32]], and iron transporter deficiency [ΔtonB [33]] have all been evaluated for their protective potential and are outlined in Table 1. Of these mutated LAVs, most notably, mice vaccinated with the auxotrophic amino acid synthesis mutant strain B. mallei (ΔilvI) demonstrated a T helper cell (Th) type 1-like immune response resulting in significant protection against a lethal aerosol challenge with B. mallei, suggesting a prominent role for Th1-skewing immune responses [27]. Similarly, Haque et al., observed significant expression of cytokines IFN-γ, IL-12, and IL-18, as early mediators for protection, against acute infection in BALB/c mice following intraperitoneal (i.p.) vaccination with a B. pseudomallei auxotrophic ilvI mutant strain (ΔilvI/2D2) [28]. When severe combined immunodeficiency (SCID) mice received adoptively transferred T cells from 2D2-boosted mice, they were significantly protected from an i.p. challenge of B. pseudomallei. This would suggest that T cells are protective against melioidosis, independent of B cells [28]. However, the vaccine failed to protect completely, since 2D2-immunized mice demonstrated initial protection with a mean survival time of 52 days but mice began to succumb to infection 30 days post-challenge, with histopathological analysis revealing the presence of splenic abscesses. Using an auxotrophic B. mallei mutant deficient in an iron active transport system (ΔtonB), vaccination of mice resulted in a significant increase in survival after an acute inhalation (i.n.) challenge with, separately, B. mallei (100%) and B. pseudomallei (75%) [33]. Moreover, serum from vaccinated mice exhibited B. mallei-specific immunoglobulin (Ig) G1, IgG2a, and IgM [33]. Since many of the virulence genes mentioned above are conserved among the Burkholderia genus, it would be interesting to evaluate their efficacy against other strains such as B. cepacia, the bacterium implicated as the causative agent of respiratory infections in patients with cystic fibrosis [34]. Infection/prevention models against B. cepacia is an area of research that has been largely ignored [35].

Table 1.

Melioidosis and glanders vaccines under pre-clinical development

Vaccine	Route of administration (vaccine/challenge)	Challenge model (strain)	Survival (%) post challengea	Reference
Live attenuated:
purN mutant (ΔpurN)	i.n./i.p.	B. pseudomallei (E8)	100% at day 9	[24]
purM mutant strain (ΔpurM/Bp82)	SubQ/i.n.	B. pseudomallei (1026b)	100% (C57BL/6) and 60% (BALB/c) at day 60	[25]
ilvI mutant (ΔilvI/2D2)	i.p./i.p.; aerosol/aerosol	B. pseudomallei (576); B. mallei (ATCC 23344)	30% at day 70; 80% at day 35; 50% at day 30	[28]; [26]; [27]
aroC mutant (ΔaroC/A2)	i.p./i.p.	B. pseudomallei (A2)	80% (C57BL/6) and 0% (BALB/c) at 5 months	[29]
asd mutant (Δasd)	i.n./i.n.	B. pseudomallei (1026b)	100% at day 80 (acute infection); 0% at day 60 (chronic infection)	[30]
relA/spT mutant (ΔrelA/spT)	i.n./i.n.	B. pseudomallei (576)	100% at day 30 (C57BL/6)	[31]
tonB/hcp1 mutant (ΔtonB/hcp1 CLH001)	i.n./i.n.	B. mallei (CSM001); B. mallei (ATCC 23344)	100% survival at day 35	[32]
tonB mutant (ΔtonB/TMM001)	i.n./i.n.	B. mallei (CSM001) & B. pseudomallei (K96243)	100% at day 28; 75% at day 36	[33]
tssN mutant strain (ΔtssN/BMAA0728)	aerosol/aerosol	B. mallei (ATCC 23344)	67% at day 21	[40]
Capsular polysaccharide (CPS)- expressing mutant B. thailandensis strain	i.p./i.p.	B. pseudomallei (K96243)	100% at day 35	[41]
Subunit:
Lipopolysaccharide (LPS)	i.p./i.p.; i.p./aerosol	B. pseudomallei (K96243); B. pseudomallei (NCTC 4845)	50% at day 35; NS at day 5	[47]; [48]
Purified outer membrane protein W (ompW)	i.p./i.p.	B. pseudomallei (576)	75% at day 21	[49]
Purified outer membrane protein 85 (omp85)	i.p./i.p;	B. pseudomallei (D286)	70% at day 15	[50]
Purified outer membrane vesicles (OMVs)	i.p./i.p.	B. pseudomallei (K96243)	67% at day 21	[53]
Purified MprA protein (SmBpF4)	i.p./i.p.	B. pseudomallei (D286)	100% at day 25	[54]
Purified LolC protein	SubQ/i.p.	B. pseudomallei (K96243)	80% at day 42	[55]
Purified PilV protein	SubQ/SubQ	B. pseudomallei (G207)	NS	[57]
Purified proteins BPSL1897, BPSL3369, BPSL2287	i.p./i.p.	B. pseudomallei (K96243)	75% at day 40	[58]
CPS and O-polysaccharide glycoconjugate (CPS2B1)	subQ/i.p.	B. pseudomallei (K96243)	90% at day 21	[59]
OPS II glycoconjugate	i.p./i.n.	B. pseudomallei (K96243)	40% at day 14	[61]
Gold nanoparticles (AuNPs) glycoconjugates (TetHc, Hcp1, FliC)	i.n./i.n.	B. mallei (ATCC 23344)	90% (TetHc; Hcp1) and 60% (FliC) at day 21	[64]
AuNP glycoconjugates (LPS-FliC)	aerosol/aerosol	B. mallei (ATCC 23344)	50% at day 80 (Rhesus macaque)	[65]
Polymer-encapsulated adjuvant and whole killed B. pseudomallei	SubQ/i.p.	B. pseudomallei (1026b)	88% at day 14	[73]
Cationic liposomes complexed with adjuvant and whole killed B. pseudomallei	i.m./i.p.	B. pseudomallei (1026b)	100% at day 30	[77]
Cationic liposomes complexed with plasmid DNA and whole killed B. pseudomallei	i.n./i.n.	B. pseudomallei (1026b)	100% at day 40	[79]

^aChallenge model is BALB/c unless otherwise indicated

Intranasal: i.n.;

Intraperitoneal: i.p.;

subcutaneous: subQ;

Intramuscular: i.m.;

NS: no survival

Bacterial secretion systems (SS) contain a number of proteins that interact with and hijack critical host proteins and pathways, allowing the pathogen to survive and propagate in the host environment [22]. Due to their prominent role in pathogenicity, SS proteins have been exploited in LAVs against Burkholderia spp. (Table 1; [36, 23, 37]). Memisevic et al., used a bioinformatics and a yeast two-hybrid approach to identify uncharacterized and novel virulence factors that were conserved between both B. mallei and B. pseudomallei [38, 39]. Using this information, they created three B. mallei mutant strains (ΔBMAA0728, ΔBMAA1865, ΔBMAA0553), one of which (ΔBMAA0728) lacks the virulence gene encoding components of a type VI SS [38]. As an LAV, this mutant strain (ΔBMAA0728/tssN) demonstrated delayed MNGC formation in vitro [40]. Vaccination of BALB/c mice lead to significant survival (67%) 21 days post-infection in an aerosol challenge model of glanders; however, 100% mortality was observed at 60 days’ post infection [40]. More recently, Hatcher et al., generated a B. mallei mutant strain deficient in both iron transport and a type VI SS protein (ΔtonB/hcp1/CLH001). Immunization with CLH001 in both BALB/c and NSG (severely immunocompromised) mice resulted in complete protection (100%) against lethal i.n. challenge with B. mallei and reduced bacterial burden after 35 days following a high dose challenge [32]. Although the prevention of the chronic infection was not evaluated, this attenuated strain demonstrated potential to protect against glanders in a pre-clinical challenge model.

LAV candidates targeting various virulence factors have also been prepared and evaluated; however, all have failed to demonstrate sterilizing immunity against long-term infections, which are typically observed 30 days post-challenge [23, 41]. In addition to the economic burden associated with designing and manufacturing LAVs, there are significant safety concerns due to the ability of attenuated pathogens to transform into virulent strains, limiting therapeutic opportunities for elderly (>65 years) and immunocompromised individuals [42]. For these reasons, it is important to consider other vaccine formulations.

Subunit vaccines

A safer alternative to LAVs is a subunit protein vaccine, which uses an antigenic bacterial component to induce an immune response. Without the use of a live bacterium, subunit vaccines are typically formulated with immune stimulating adjuvants to boost cellular and humoral immunity. Several subunit vaccines against Burkholderia have been evaluated, and reviewed extensively [43, 23, 35, 44]. Secreted and membrane-associated proteins as well as polysaccharides are often used as antigens in subunit vaccines because they are abundantly expressed by gram-negative bacteria and are shown to be potent stimulators of host immune responses. For Burkholderia subunit vaccines, these include lipopolysaccharides (LPS) and outer membrane proteins and vesicles (OMP and OMV, respectively). LPS have shown to play an important role in the pathogenesis of Burkholderia infection and a number of LPS molecules have been identified in B. pseudomallei and B. mallei, which are therefore, a likely target for subunit vaccines [45]. As a potent inducer of host immune responses, LPS O-antigen has been shown to provide epitopes for the development of antigen-specific antibodies, and the lipid A region is a potent agonist for Toll-like receptors (TLRs) [46]. In an animal model for melioidosis, i.p. immunization with LPS isolated from B. pseudomallei induced predominantly IgM and IgG3 responses and afforded significant protection (50% at day 35) following an i.p. challenge with a lethal dose of B. pseudomallei [47]. This protection; however, was not observed in an aerosol challenge [47, 48]. Additionally, passive immunity was observed and provided protection (mean time of death- 29 days) following an i.p challenge of B. pseudomallei [48].

Studies have also investigated the immunogenic potential of B. pseudomallei-specific OMPs as a subunit antigen against Burkholderia infections. Casey et al., evaluated the surface protein ompW, which functions in the attachment of bacterium to host epithelial lining, as a subunit antigen because it is conserved among several Burkholderia spp. [49]. Purified ompW administered via i.p. injection with the adjuvant monophosphoryl lipid A (MPL) induced a potent Th1-skewing serological response and protected BALB/c mice (75% survival up to 21 days) against lethal i.p. challenge of B. pseudomallei; however, failed to surpass the efficacy of the live attenuated 2D2 vaccine control [49]. Using a different adjuvant, vaccination with recombinant omp85 antigen emulsified in Freund’s complete adjuvant (CFA) triggered a Th2-type immune response resulting in reduced bacterial loads in organs (lung, liver and spleen) and 70% survival up to 15 days after lethal i.p. B. pseudomallei challenge [50]. Additionally, mouse sera from the rOMP85 immunization group promoted complement-mediated killing and opsonization of B. pseudomallei by human polymorphonuclear cells [50]. OMVs are secreted by bacteria and are enriched with multiple proteins, lipids, and polysaccharides and have demonstrated robust immunogenicity as vaccine antigens against lethal i.n. and i.p. Burkholderia challenges when administered with an aluminum hydroxide (alum) adjuvant [51, 52]. Intraperitoneal vaccination of B. pseudomallei-derived OMVs afforded significant protection (67% at 21 days) against acute septicemic B. pseudomallei infection (i.p. challenge) and induced robust OMV-, LPS-, and CPS-specific serum IgG and IgM antibody responses in mice [53]. Similarly, B. pseudomallei-derived OMVs administered by subcutaneous injection, along with an adjuvant (CpG oligodeoxynucleotides; TLR 9 agonist), in a non-human primate model (rhesus macaque) also resulted in the production of OMV-, LPS-, and CMP-specific IgG serotypes [52]. Since members of the Burkholderia genus express structurally similar polysaccharides and membrane proteins, CPS’s, LPS’s, OMP’s, and OMV’s all provide promising antigens that can be used as a multivalent vaccine strategy against melioidosis.

Other approaches to identify or isolate protein antigens from B. pseudomallei have also been employed and are outlined in Table 1 [54]. Notably, immunization with LolC (ATP-binding cassette (ABC) transporter-type lipoprotein-releasing protein) demonstrated significant humoral and cellular immunogenicity that resulted in 80% survival at 42 days when administered with the adjuvant MPL [55]. Additional antigens, such as FliC (the flagella major subunit), have been proposed as a potential subunit vaccine candidate due to its ability to elicit long-lasting immune memory [56]. Vaccination with PilV (type IV pilin protein) and the adjuvant CFA produced high levels of IgG, strongly biased toward IgG1, but was unable to protect BALB/c mice against lethal i.p. B. pseudomallei challenge following subcutaneous immunization [57]. More recently, Champion et al., identified a series of genes: BPSL1897 (uncharacterized protein), BPSL3369 (acetaldehyde dehydrogenase), and BPSL2287 (iron cluster assembly protein) expressed by the bacteria colonizing the spleens and lungs of chronically infected mice [58]. Following co-administration with these recombinant proteins and MPL, significant protection was observed for 42 days against lethal challenge [58].

In addition to protein-based subunit antigens, glycol-conjugate-based vaccines, in which bacterial surface polysaccharides are chemically conjugated to a carrier protein, have been demonstrated to be highly effective subunit vaccines against Burkholderia. For example, type O-polysaccharide and CPS antigens covalently linked to carrier proteins demonstrated high IgG titers and protected mice from a lethal i.p. B. pseudomallei challenge [59, 60]. Furthermore, it was demonstrated that mice co-administrated with a capsular polysaccharide (CPS) glycoconjugate and the promising subunit antigen, LolC, exhibited higher survival rates following lethal challenge compared to single administration [59]. Although promising, many of the current glycoconjugate formulations do not include glycans from the native host since B. pseudomallei requires BSL3 facilities, limiting their accessibility. To address this hurdle, Garcia-Quintanilla et al. utilized a novel method for synthesizing glycoconjugates through exploitation of the protein glycosylation machineries of bacteria; however, purified OPS II glycoconjugate provided only partial protection (40% at day 12) against lethal i.n. challenge of B. pseudomallei [61]. Overall, subunit vaccines, although safer than LAVs, demonstrate only partial protection against Burkholderia spp. infections and unfortunately remain stagnant in an attempt at pre-clinical testing, with relatively little evaluation against glanders.

Micro- and nano-systems for improved vaccine formulations against Burkholderia

Advances in nanotechnology have provided a significant impact in vaccine development, particularly in the use of metal-, polymer- and lipid-based nanoparticles and nanoemulsions as formulations to enhance vaccine efficacy [62]. Gold nanoparticles (AuNPs) are promising candidates for biological applications due their unique physicochemical properties, biocompatibility, and ease of synthesis and conjugation [63]. In a vaccine model against glanders, AuNPs covalently coupled with protein antigens (TetHc, Hcp1 and FliC) and conjugated LPS (isolated from B. thailandensis) generated significantly higher antibody titers compared to LPS alone and resulted in improved, but incomplete protection against a lethal i.n. B. mallei challenge [64]. A similar study evaluated the protective efficacy of AuNP glycoconjugates (LPS- FliC) in a non-human primate model (rhesus macaque) using an aerosol B. mallei challenge. Vaccination with the AuNP formulation demonstrated protection and LPS-specific IgG titers on day 80 in three out of six vaccinated animals that survived [65].

Polymeric microparticle (MP) carriers, which can encapsulate multiple adjuvant and antigen components, provide many advantages over metal-based nanosystems because they are biodegradable, can be sized to passively target antigen presenting cells (APCs), and can serve as an immune depot. Vaccine carriers composed of poly-(lactide-co-glycolide) (PLGA) have been extensively characterized in animal models and are currently being evaluated in Phase I clinical trials (Selecta); however, they have not been utilized for vaccines against Burkholderia [66]. Our group utilizes the acid sensitive biopolymer, acetalated dextran (Ac-DEX), because it is immunologically inert, has triggered release in the low pH environments of the phagosome and lysosome, and can be formulated with tunable degradation for formation of a vaccine depot [67–70]. Ac-DEX MPs have been previously shown to enhance cross-presentation of subunit antigens leading to protection in an anthrax vaccine model [71, 72]. In a model against acute melioidosis, BALB/c mice were vaccinated subcutaneously with free whole killed B. pseudomallei cell lysate (antigen) and Ac-DEX MPs encapsulating the adjuvant resiquimod (TLR 7/8 agonist) [73]. One of the two studies presented followed a rapid immunization/challenge schedule (vaccination on day 0, boost on day 7, followed by challenge on day 14). Resiquimod MPs co-delivered with soluble lysate as the antigen elicited robust Th1- and Th2-type immune responses leading to significant delay to time of death (88% at day 14) compared to empty MP-treated mice when given a lethal i.p. B. pseudomallei challenge; however, only 16% of mice survived past 26 days post-challenge [73]. Additionally, B. pseudomallei was not detected in the liver and blood of several surviving mice (2/4) following lethal challenge in the lysate with resiquimod MP group. Moreover, a fraction of other surviving mice in all the groups vaccinated with encapsulated resiquimod (resiquimod MPs, lysate MPs co-delivered with resiquimod MPs, and resiquimod MPs with lysate absorbed on alum) had sterile blood and livers (~10% of total mice challenged in the group), with some mice in the alum and resiquimod MP group having sterile spleens. This suggests that a sterilizing immunity was achieved in a fraction of immunized mice. When this platform was expanded to a more traditional day 0 and 14 prime and boost with a 28-day challenge, somewhat similar trends in survival were observed, indicating that perhaps these formulations could be used effectively in a more rapid timeframe, such as post-exposure prophylaxis [73]. Although the vaccine lead to incomplete protection, likely due to lack of discrete antigens, Ac-DEX represents a novel delivery vehicle for a subunit vaccine against melioidosis.

Nanoemulsions (NE) are a clinically relevant adjuvant system that could be an alternative to alum for many subunit vaccines. Recently, the nanoemulsions MF59, which is a squalene-based oil-in water emulsion, was FDA-approved as an adjuvant for influenza vaccines (FLUAD, Novartis) [74]. The nanoscale size and the negative charge of the NE-antigen mixture allows the vaccine to penetrate mucosal layers and to facilitate cellular uptake, which makes it an ideal vehicle for vaccine carriers against aerosolized Burkholderia infections. Although NEs have not been used for B. pseudomallei or B. mallei vaccines, Makidon et al., co-administered a NE with the OMP antigen (OmpA) derived from B. cepacia and found that immunized mice were protected against pulmonary bacterial colonization after a B. cenocepacia or B. multivorans challenge [74]. Moreover, vaccination with the NE adjuvanted vaccine yielded a balanced Th1/Th2 cellular response and production of mucosal IgA [75].

Liposomes, a lipid bi-layer vessel, have been used as adjuvant complexes in Burkholderia vaccines. When used in a melioidosis vaccine, whole-killed B. pseudomallei administered with cationic liposomes ((2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium; DOTAP) ionically complexed with immunomodulatory CpG afforded complete protection (100%) for up to 30 days against a lethal B. pseudomallei challenge [76]. CpG is a synthetic oligodeoxynucleotide adjuvant that has been shown to promote a TLR9-mediated Th1 immune response when used in several bacterial vaccines [77, 78]. Henderson et al., evaluated the use of cationic liposomes complexed with non-coding plasmid DNA (CLDC) as a potential mucosal vaccine adjuvant [79]. Immunization with plasmid and heat-killed B. pseudomallei induced efficient uptake by DCs, potent mucosal IgA antibody responses, sustained antigen-specific CD8+ T cell responses, and afforded 100% protection (>40 days) from a lethal i.n. B. pseudomallei challenge [79].

Combining antigen delivery (e.g., nanoparticles, biopolymers, liposomes) with potent adjuvant stimulation represent promising vaccine models against melioidosis, specifically in their ability to more efficiently deliver vaccine components and generate a broader immune response.

Conclusions

Despite the many strategies explored in the development of a vaccine against Burkholderia, all have failed to induce complete and sterilizing immunity over an extended period of time. Most of the reported studies observed short-term survival followed by death in response to a lethal challenge of B. pseudomallei or B. mallei (Table 1). Although no studies demonstrated complete sterilizing immunity, several promising LAV and subunit vaccines against Burkholderia have been proposed. Most notably, a mutant strain deficient in iron transport and a type IV SS [ΔtonB/hcp1) [32]] and several mutant strains lacking functional virulence factor genes [39, 40] demonstrated significant protection against a lethal Burkholderia challenge. Although LAVs are potent stimulators of immunity, safety concerns make them unlikely candidates for clinical applications; therefore, subunit vaccines become more practical. However, subunit vaccines are poorly immunogenic and mostly require co-administration with an adjuvant. LolC, the leading subunit Burkholderia antigen to date, conferred significant protection when administered with the adjuvant, MPL [55]. Promising advances in vaccine development have utilized novel formulations involving emulsions, polymeric particles, liposomes, and metal-based nanoparticles. A notable vaccine candidate uses the cationic liposomal formulation with a plasmid ionically absorbed to the surface to serve as an adjuvant. When co-administered with heat-killed B. pseudomallei, a strong CD8+ immune response was generated and 100% survival past 40 days was reported following an i.n. B. pseudomallei challenge [79]. However, future work needs to be done in this field as only a handful of nano and micro-particle formulated vaccine candidates have been reported.

For vaccine candidates to progress beyond pre-clinical stages of development, standardization in experimental design needs to be taken into account. Inconsistency across studies has prevented researchers from moving forward in deciding candidate vaccines for evaluation in non-human primates, in addition to the uncertainty regarding which NHP model is best for Burkholderia infection [80]. Route of vaccine administration and bacterial challenge seem to be major factors influencing protective immunity against Burkholderia infections. While the i.p. route is used most commonly in experimental mouse models because of its ease of administration, aerosol, i.n., or subcutaneous challenges could be considered a more physiologically relevant model for melioidosis. An aerosol challenge is certainly deemed a more rigorous challenge route; however, in addition to variable dosing, it requires specialized equipment at a BSL3 facility, which significantly restricts availability. The variable dose of a variety of bacterial strains also makes it difficult to compare studies. For example, it has been reported that a B. pseudomallei challenge dose required for a rapidly fatal infection is significantly higher via the subcutaneous route compared to the inhalation route [81]. A careful evaluation and reporting of LD₅₀’s for each challenge strain and route can help to mitigate these differences. Moreover, relevant animal models need to be taken into account when evaluating vaccine efficacy. Most common strains of mice reported in studies are BALB/c and C57BL/6, which have demonstrated differential susceptibility to B. pseudomallei infection, with C57BL/6 being more resistant [82]. Since BALB/c mice are classically a model of acute infection, with C57BL/6 modeling more chronic infection, evaluation in both strains would be warranted in a rigorous vaccine study. Additionally, no studies have evaluated vaccine efficacy in models with underlying conditions such as diabetes and immunosuppression, which make up a large population of susceptible individuals in endemic regions and would certainly alter vaccine efficacy.

Because the field of Burkholderia vaccines has not advanced much beyond preliminary studies looking at vaccine efficacy, it has been difficult to thoroughly evaluate the mechanism of immune responses. Studies into CD8+ and CD4+ T cell response are not well reported because immunological tools like immunodominant peptides or tetramers have yet to be resolved for any of the antigens prepared. Perhaps a more in-depth analysis of epitopes could provide knowledge that will allow researchers to design better antigens that improve immunogenicity, with the potential to provide protection over the course of long-term infection. The work of Champion et al., evaluating expression markers in the bacteria of chronically infected mice [58] is a step in the right direction to create vaccines to combat long-term infection, approaching the problem as an immunologist rather than a microbiologist could further lead new information on vaccine improvement. A collaborative effort from microbiologists and immunologists, as well as vaccine formulation scientists, could work to overcome this difficult-to-prevent infection. By uniting these areas of knowledge, research efforts should be able to focus on developing a broad-spectrum vaccine that confers long-lasting immunity to multiple strains of Burkholderia responsible for lethal melioidosis and glanders.