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

Abstract

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.

1. Introduction

Melioidosis is a multifaceted human disease, mainly endemic in Northern Australia and Southeast Asia, with increased case reports in other parts of Asia, Central and South America, the Caribbean, and Africa [1–5]. It has been calculated that the disease has an estimated incidence of 165,000 cases per year [4,6], and the mortality rate can be over 50% worldwide [3,4,6,7]. Further, this disease is prevalent in many countries where under-reporting of melioidosis cases is a major issue, which, along with a lack of both appropriate clinical and laboratory expertise and microbiological services, complicates its identification [4,8].

Melioidosis is an infectious disease caused by Burkholderia pseudomallei, a pathogen whose main infection routes are inhalation (aerosolized environmental bacteria), percutaneous inoculation (through an open wound), and ingestion (contaminated food or water) [3,8,9]. Disease can manifest as acute, leading to fatal disease, latent and/or chronic infections, due to the pathogen’s ability to survive intracellularly [8–10]. The symptoms of melioidosis vary depending on the infection route, but its main clinical presentation is pneumonia, followed by genitourinary and skin infections, bacteremia, osteomyelitis, and neurological manifestations [7–10]. In an acute infection, it has been reported that patients can develop sepsis with or without pneumonia or skin abscesses, independently of infection route [8]. The dearth of nonspecific symptoms and its similarity with many other diseases, especially tuberculosis, has gained melioidosis in the name of ‘the great mimicker’ [3,8]. Melioidosis is associated with high mortality rates, but the disease is also complicated by comorbidities and risk factors present in about 80% of the patients, such as diabetes mellitus (more than 50%), hazardous alcohol consumption, chronic lung or renal diseases, and thalassemia, among others [4,6–8,11–13]. A recent study demonstrated a higher frequency of this disease in men as well, and different immunoserological markers have also been identified between men and women in diabetes mellitus patients [14].

B. pseudomallei is a Gram-negative facultative-intracellular bacillus that is intrinsically resistant to a wide range of clinically-used antibiotics [3,7,15,16]. A recommended antibiotic treatment includes 2–8 weeks of intravenous dosing followed by 3–6 months of oral treatment, which targets the intracellular stage of the pathogen that is associated with bacterial persistence to eradicate remaining bacteria [13,17]. Even though the oral treatment phase increases the chances of the patient to clear the infection and reduces the rate of relapse, chronic infection can still occur.

B. pseudomallei is classified by the U.S. Centers for Disease Control (CDC) as a Tier 1 Select Agent for its biothreat potential [1,2,8,18–20]. Both the growing worldwide endemicity of the pathogen and its potential use as a biothreat agent, make vaccination an appropriate strategy to ensure the disease does not become an increased public health concern. The main obstacles to overcome for the development of an effective vaccine are the ability of the bacteria to evade the immune system while surviving in the intracellular environment, as well as the phenotypic variability among virulent endemic strains of B. pseudomallei [13,21,22].

Due to the need to develop an effective vaccine against melioidosis, paired with difficulties in comparing studies due to differences in vaccination protocols, bacterial strains, and animal models (among others), an independent group of scientists was created in 2013 in an attempt to accelerate the development of an effective treatment called the Steering Group on Melioidosis Vaccine Development (SGMVD) [22]. It was noted that many of the melioidosis vaccine studies performed in the past decade used C57BL/6 and BALB/c mice, which are prototypical Th1- and Th2-type mouse strains, respectively [23]. BALB/c mice represent an acute infection model of melioidosis and are suitable to study virulence, whereas C57BL/6 are more resistant to infection and are a reliable model to study chronic infection [24]. Further, C57BL/6 mice are also currently used for melioidosis vaccine screening [23]. Several vaccines have been developed through the years, and excellent reviews have been published elsewhere [11,23,25], we have decided to focus on a few vaccines that are considered the leading candidates currently funded by either the U.S. Department of Defense (DOD) [21,26–29] or by the U.S. National Institute of Health (NIH) [30]. Such vaccines include live-attenuated strains [27–29], gold-nanoparticle antigen combinations [30], subunit glycoconjugates [21], and outer-membrane vesicles [7,26,31] (Figure 1).

Figure 1. The leading B. pseudomallei vaccine candidates.

A) Glycoconjugate: CPS coupled to CRM197 carrier protein tested alone and in combination with Hcp1 or TssM recombinant protein, B) Live-attenuated mutant strains: ΔtonB Δhcp1 and ΔhisF, C) OMVs and D) Gold-nanoparticle: AuNP-FlgL-LPS, AuNP-hemagglutinin-LPS, AuNP-Hcp1-LPS or in combination (combo).

Live-attenuated vaccines are mutated strains designed to maintain immunogenicity but with significantly reduced virulence. This has been an effective approach for obtaining complete protection against many human pathogens, although not all attenuated strains are able to induce protective immunity [11,32]. Furthermore, Outer-Membrane Vesicles (OMVs) have been developed and tested against B. mallei and B. pseudomallei infection [11,23]. OMVs are non-replicating nanovesicles secreted by Gram-negative bacteria that are enriched in biologically active proteins including LPS, microbial lipids, and other potential immunogens [26,33,34].

Subunit vaccines are another attractive, safe alternative to live-attenuated vaccines [23,32]. In this context, immunological protection of several potential vaccines has been evaluated, many of which use conserved antigens related to virulence-related properties [21,35].

Lastly, a more recent, innovative approach is the development of multicomponent nanovaccines, which are an efficient platform that enable the enhancement of immune responses while maintaining a good safety profile [30]. Gold nanoparticle glycoconjugates have been previously reported to provide significant protection in murine and non-human primate models of inhalational glanders, a disease caused by Burkholderia mallei [36,37], and also against murine melioidosis [30].

1.1. Live-attenuated vaccines

1.1. A. pseudomallei ΔtonB Δhcp1 (PBK001)

The double gene deletion mutation of the tonB and hcp1 genes was originally constructed in B. mallei ATCC 23344 (vaccine strain name CLH001) [38], and a similar deletion approach was used to create the same mutations in B. pseudomallei K96243 (vaccine strain name PBK001) [28]. The first deleted gene, tonB, encodes a protein involved in the uptake of iron [39]. TonB is a periplasmic protein tethered to the inner-membrane, which forms a subunit complex with ExbB and ExbD, together known as TonB-dependent transporters [39]. The iron uptake mechanism of Burkholderia is proposed to be similar to other Gram-negative bacteria [40]. For example, the Burkholderia siderophore (ferric-ornibactin) is recognized by TonB-dependent receptors, and the TonB complex (TonB-ExbB-ExbD) is used as a proton motive force energizer of siderophore translocation into the periplasm [39]. The second mutated gene, hcp1 (hemolysin co-regulated protein 1), is an essential protein component required for the assembly and functionality of the type 6 secretory system cluster 1 (T6SS-1). Hcp1 forms hexameric rings that are part of the building blocks of the bacterial injectosome resembling the tails of bacteriophages [41]. The T6SS-1 has been characterized as a major virulence determinant and plays an important role in the intracellular lifestyle of B. pseudomallei. The T6SS-1 participates in the formation of Multinucleated Giant Cells, thereby allowing bacterial spread from cell to cell, while limiting host immune detection [42,43]. The B. pseudomallei tonB isogenic mutation was created using the pMo130ΔtonB suicide plasmid, which was introduced into the recipient wild type strain, creating an allelic exchange. A similar approach was used to create the hcp1 mutation [28]. The double mutation (ΔtonBΔhcp1) was constructed ‘unmarked’ without the introduction of any antibiotic resistance cassette and has been fully characterized at the genetic level. The introduction of a double deletion mutation in the bacteria significantly reduces the likelihood of reversion to a wild-type phenotype and increases the safety of the vaccine.

B. pseudomallei ΔtonBΔhcp1 (PBK001) is a significantly attenuated strain when compared to the parental strain (B. pseudomallei K96243) using same inoculation route, mouse strain, and lethal time window. Intranasal (i.n.) dosage of 1.5 × 10⁴ CFU of PBK001 was equivalent to 10 LD₅₀ of B. pseudomallei K96243 when using the C57BL/6 mouse model of infection at day 21 post infection. An intranasal prime and two boosts immunization with the equivalent dose of PBK001 does not cause disease in mice, and experimental evidence demonstrated that the vaccine strain is cleared from the lungs of immunized animals within 2 days after its administration, at which point no bacteria were found in any of the targeted organs (liver and spleen). Recently, PBK001 has been approved by the CDC to be excluded from the select agent register.

C57BL/6 mice receiving the PBK001 vaccine have shown full protection against B. pseudomallei K96243 up to 4 weeks post-aerosol challenge (7–12 LD₅₀) (Table 1). Five out of seven mice (71%) receiving the vaccine showed no countable bacteria in the lungs, liver, and spleen, and less than 20 CFU/organ were detected in 2 out of 7 (29%) animals. The PBK001 vaccine strongly induced a humoral immune response by producing high titers of B. pseudomallei-specific IgG antibodies and its subclasses. The IgG2a/IgG1 ratio indicated a Th1-biased immune response, and specific Th1 (IFN-γ) and Th17 (IL-17A) cytokines were produced in response to PBK001 vaccination. Vaccine-induced antibodies were suggested to be the primary correlate of protection. However, it was observed that the absence of CD4⁺ or CD8⁺ T cells, due to T cell depletion in PBK001 vaccinated mice, did not affect the protective phenotype [28].

Table 1.

Summary of the leading B. pseudomallei vaccine candidates funded by US agencies.

Immunization					Challenge
Vaccine type	Antigens/mutants	Adjuvant(s)	Route	Mouse Strain	Strain	Route	Dose (CFU/ mouse)	Survival	Antibody correlates protection	Reference
Live-attenuated	Bpm K96243 ΔtonB Δhcp1	-	i.n.	C57BL/6	K96243	aerosol	1.7 × 103	100% at day 27	Yes	[28]
	Bpm MSHR668 ΔhisF	-	s.c.	BALB/c	K96243	i.p.	3.1 × 106	100% at day 25 50% at day 60	No	[29]
Nanoparticle	AuNP-combo-LPS	Alhydrogel + poly(l:C)	s.c.	C57BL/6	K96243	i.n.	5.3 × 104	100% at day 35	No	[30]
	AuNP-FlgL-LPS							90% at day 35
	AuNP- hemagglutinin- LPS							20% at day 35
	AuNP-Hcp1-LPS							10% at day 35
Glycoconjugate subunit	CPS-Hcpi	Alhydrogel + CpG	s.c.	C57BL/6	K96243	aerosol	1.54 × 103	100% at day 35	Yes	[21]
	CPS-TssM							80% at day 35
	CPS-CRM197							67% at day 35
Outer-Membrane Vesicle	OMVs from Bpm 1026b	-	i.n. s.c.	BALB/c	1026b	aerosol	1 ×103	20% at day 14 60% at day 14	Yes	[34]
	OMVs from Bpm 1026b	-	s.c.	BALB/c	K96243	i.p.	2 × 104 8 × 105	100% at day 21 67% at day 21		[26]

LPS, lipopolysaccharide; CPS, capsular polysaccharide; CpG, cytosine-phosphate-guanine; OMV, outer-membrane vesicle; i.n., intranasal; s.c., subcutaneous; i.p., intraperitoneal.

Likewise, the B. mallei ΔtonBΔhcp1 double mutant vaccine strain, known as CLH001, afforded protection to the majority of C57BL/6 mice (87.5%) against lethal aerosol challenge with B. pseudomallei K96243, and 50% (3 out of 6) of the surviving mice showed bacterial clearance in the lungs, liver, and spleen four weeks post challenge [27].

1.1. B. pseudomallei 668 ΔhisF

This attenuated B. pseudomallei auxotroph was constructed by deleting a 65-bp region of the hisF gene in the highly pathogenic B. pseudomallei MSHR668 parental strain to create the vaccine candidate 668 ΔhisF [29]. The deletion of hisF gene impaired cellular protein biosynthesis and de novo purine biosynthesis processes in these intracellular bacteria [44].

The 668 ΔhisF vaccine showed a highly attenuated phenotype in immunocompromised NOD/SCID mice. To determine the degree of attenuation for the 668 ΔhisF vaccine strain, different doses of parent strain (wild-type MSHR668) were compared using the same inoculation route, mouse strain, and time course. Intraperitoneal injection (i.p.) of 1.02 × 10³ CFU of 668 ΔhisF, which was equivalent to 79 LD₅₀ of the wild-type MSHR668 strain, did not have an effect on survival of the immune defective mice and nearly all (90%) of their spleens and livers were found to be sterile. In the protection study, BALB/c mice were subcutaneously (s.c.) vaccinated with a prime and a boost, 3 weeks apart, of 668 ΔhisF, following by an intraperitoneal challenged with 50 LD₅₀ of B. pseudomallei K96243. Vaccination with the 668 ΔhisF protected BALB/c mice during the acute phase (100% survival at day 25 post-infection) and chronic phase of infection (50% survival through 60 days post-infection) (Table 1). Interestingly, the investigators found that the total IgG, IgG2a, and IgG1 antibody responses generated by this mutant did not correlate with protection. However, significantly elevated expression of IFN-γ was observed in the vaccination group after ex vivo re-stimulation of splenocytes and was closely correlated to the protective phenotype [29].

1.2. Nano-particle vaccines

Multicomponent nanovaccines are efficient platforms that help enhance immune responses while maintaining a good safety profile. The gold nanoparticle (AuNP) glycoconjugates were first reported to provide significant protection in murine and non-human primate (NHP) models of inhalational glanders [36,37]. In a recent study by Muruato and colleagues, they identified B. pseudomallei antigens that were further incorporated into the nanovaccine candidates [30]. Incorporation of these proteins to the AuNP glycoconjugate vaccines was optimized and evaluated for immunogenicity in a murine model of melioidosis. The potential Burkholderia vaccine antigens were identified using an in silico (bio- and immuno-informatics) reverse vaccinology approach. Seven predicted immunogenic candidates (Hcp1, FlgD and L, Hemagglutinin, and three porins) were identified, and four of these novel proteins (OmpW, FlgD, hypothetical porin, and FlgL) were confirmed to be seroreactive against sera taken from chronically infected mice and sera from convalescent human melioidosis patients. Three of these candidates, including hemagglutinin, Hcp1 and FlgL, were individually conjugated into the AuNP glycoconjugate, and the lipopolysaccharide (this LPS is immunogenic but non-toxic) purified from Burkholderia thailandensis was also incorporated into the formulation [30]. The AuNP vaccine formulations were used alone or in combination (combo) to immunize C57BL/6 mice by the s.c. route using a prime and two boosts vaccination schemes. After the final vaccination, the immunized mice were i.n. challenged with 3.4 LD₅₀ of B. pseudomallei K96243. It was found that mice receiving AuNP-FlgL-LPS and AuNP-combo-LPS showed 90% and 100% protection at day 35 post challenge, respectively, whereas other groups demonstrated 20% survival (adjuvant only and AuNP-hemagglutinin-LPS), 10% survival (LPS alone and AuNP-Hcp1-LPS), and 0% survival (AuNP-BSA-LPS) [30] (Table 1). Moreover, a significant reduction of bacterial colonization in the lungs, liver, and spleen in the AuNP-FlgL-LPS and AuNP-combo-LPS vaccinated groups was observed when compared to those receiving adjuvant alone or other immunized groups. Even though immunized mice generated high protein- and LPS-specific IgG titers, this study was unable to correlate protection with high antibody titers.

Recently, further immunizations have been performed using additional antigens, and different routes of immunization have been tested against B. pseudomallei infection (unpublished data). The other proteins, along with LPS from B. thailandensis, were individually conjugated to AuNPs and used to immunize C57BL/6 mice by either the s.c. or the i.n. routes. Immunized mice that received vaccination with two porins, or a combination of these two, displayed different levels of protection with the highest degree of protection obtained from the combination formulation against both B. pseudomallei and B. mallei (unpublished data). In addition, antibody responses from the surviving mice showed strong protein- and LPS-specific IgG titers compared to naïve and control groups. Overall, these studies provide further basis for the rational identification and construction of multicomponent vaccine platforms against B. pseudomallei, B. mallei or other pathogenic Burkholderia infections such as the B. cepacia complex.

1.3. Glycoconjugate subunit vaccine

Bacterial surface polysaccharides could be potentially good antigens for vaccine formulation; however, polysaccharides are poor immunogens due to a lack of T cell involvement. Therefore, conjugation to proteins is needed to increase their immunogenicity [11]. A recent study has shown the potential use of capsular polysaccharide (CPS) in combination with TssM and Hcp1 proteins, to generate effective glycoconjugate vaccines [21]. TssM and Hcp1 are two virulence-related antigens from B. pseudomallei, which are associated with secretion systems 2 (T2SS) [45] and 6 (T6SS) [43], respectively. This study also included recombinant CRM197 as a protein carrier, which is a diphtheria toxin mutant derivative, widely used for its versatility in glycoconjugate vaccines [21,46]. The authors of the study have previously shown the protective activity of capsular polysaccharide glycoconjugates using a BALB/c mouse infection model [47].

To construct the glycoconjugate vaccine, the CPS was purified from the B. pseudomallei strain RR2683 by centrifugation followed by a hot aqueous phenol phase procedure. For coupling the CPS and CRM197 carrier protein, a conjugation strategy with sodium-meta-periodate and sodium cyanoborohydride was used. Further, the recombinant proteins Hcp1 and TssM were purified from E. coli TOP10 (pMB1001) and coupled to the glycoconjugate vaccine [21].

For the in vivo experiment, C57BL/6 female mice were subcutaneously immunized with different formulations and with the addition of the adjuvant CpG-ODN, at 0, 21, and 35 days. The vaccine groups included CPS-CRM197 (2.5 μg/dose), Hcp1 (5 μg/dose), TssM (5 μg/dose), CPS-CRM197 plus Hcp1 and CPS-CRM197 plus TssM (same doses as for each component alone), or adjuvant only (500 μg/dose of Alhydrogel + 20 μg/dose of CpG). Five weeks after the final boost, the immunized mice were challenged by aerosol with 1.54 × 10³ CFU/mouse of B. pseudomallei K96243, and the animals were monitored for weight loss and survival for 35 days. Increased survival rates were obtained in the glycoconjugate-immunized mice: 100% for mice receiving CPS-CRM197 plus Hcp1, 80% for CPS-CRM197 plus TssM, and 67% for CPS-CRM197 alone. Only 30% (Hcp1) and 20% (TssM) of mice immunized with recombinant proteins survived (Table 1). Spleens from all glycoconjugate formulations immunized mice had no detectable bacterial load, and 70% of CPS-CRM197 plus Hcp1 immunized mice had no bacteria in their lungs or livers. All bacteria-free organs showed a normal gross pathology similar to healthy tissue, which indicated that sterilizing immunity might be able to be achieved with this vaccine after an acute inhalation challenge of B. pseudomallei [21].

Murine antibody titers were measured, and spleens were harvested to perform re-stimulations of splenocyte suspensions and to quantify IFN-γ-secreting T cells in vitro. All conjugate combinations stimulated the production of high IgM titer responses, and total IgG response against CPS was also observed. Furthermore, recombinant proteins, alone or coupled to the conjugate were enough to stimulate the production of high titers of IgM, and a total IgG response toward Hcp1 and TssM was also observed. Splenocytes from all conjugate-immunized mice as well as recombinant protein-immunized samples exhibited robust IFN-γ-secreting T cell responses after re-stimulating with each antigen (lower quantities for the CPS-CRM197 plus TssM combination) compared with mice that received adjuvant only [21].

In addition, B. pseudomallei K96243 cells were incubated with the glycoconjugate vaccine sera at a 1% concentration and performed infection of RAW 264.7 murine macrophage cells. The in vitro bacterial uptake by macrophages was promoted by CPS-specific antibodies, both alone and in combination with protein antibodies. Although the results are encouraging, a question that remains to be answered is how B. pseudomallei can induce different antibody classes and subclasses in response to these antigens during infection. Pumpuang and colleagues recently analyzed sera antibodies in healthy donors versus melioidosis patients to find new immuno-reactive candidates and identify new potential diagnostic antigens [14]. The results obtained by Burtnick and colleagues with the melioidosis glycoconjugate vaccine [21] correlated with the data where enhanced Hcp1-specific IgG and IgM titers were found in melioidosis patients as compared with healthy donors.

1.4. Outer-membrane vesicle (OMV) vaccine

A study has evaluated the protective efficacy of the acellular OMV vaccine against septicemic melioidosis caused by heterologous strains of B. pseudomallei [26]. These authors have previously tested their vaccine for its ability to confer protection against B. pseudomallei infections in inhalational murine models of pulmonary disease [34]. The authors obtained OMVs from B. pseudomallei strain 1026b by growing bacteria in LB broth until the late log phase. Then, cultures were centrifuged and OMVs from the supernatant were precipitated with ammonium sulfate and harvested again by centrifugation. Finally, trichloroacetic acid precipitation and ultracentrifugation were used for vesicle purification.

The OMV vaccine from B. pseudomallei strain 1026b was tested in BALB/c mice using either a subcutaneous (2.5 μg in 100 μl of saline) or intranasal (2.5 μg of 7.5 μl/nostril) route with a primed (day 0) and two boost (days 21 and 42) regimen [34]. Mice were challenged with 1 × 10³ CFU/mouse (5 LD₅₀) of B. pseudomallei 1026b via aerosol 1 month after the last boost. On day 14 post-infection, 60% and 20% survival were found in mice that received OMV via the s.c. and i.n. route, respectively (Table 1). In contrast, naïve mice showed 100% mortality by day 7 post-challenge. Surviving mice immunized with OMV demonstrated an absence of detectable bacteria in the lungs by day 14 post-challenge. Subcutaneously vaccinated mice showed low bacterial burden in the liver (<30 CFU), and 3 × 10³ CFU were found in 1/3 of the spleens. Mouse serum was analyzed a month after the last immunization, and high titers of OMV-specific antibodies were found with both vaccination routes. The IgG1/IgG2a ratio of the s.c. and i.n. route was equal to 7.5 and 12.2, respectively, indicating a Th2-type antibody response. The re-stimulation of T cells from mice immunized by these routes showed significantly higher production of IFN-γ compared to the control group. This result indicated a memory T cell response was induced by OMV vaccination [34].

In 2014, Nieves and colleagues performed the OMV vaccine study against heterologous B. pseudomallei strain K96243 challenge [26]. For vaccination and subsequent challenge, BALB/c mice were subcutaneously immunized at day 0, and boosted at days 21 and 42, with 5 μg of OMVs from strain 1026b in 100 μl total volume. Five weeks after the last immunization, mice were challenged intraperitoneally with 2 × 10⁴ CFU (5 LD₅₀) or 8 × 10⁵ CFU (200 LD₅₀) of B. pseudomallei K96243 strain, and survival rates were evaluated for 21 days post-infection. All the control (saline only) mice challenged with 8 × 10⁵ CFU died within 24 h, while the immunized mice with the heterologous OMVs presented 94% survival at day 7, 83% at day 14, and 67% at day 21 post-challenge. OMV vaccinated mice that were challenged with 2 × 10⁴ CFU showed 100% survival at day 21 post-challenge (Table 1). The significantly increased survival rates against septicemic B. pseudomallei infection, suggested that protection was achieved due to the treatment. However, up to 10⁸ CFU were found in the mouse spleens, indicating that sterilizing immunity was not achieved due to the bacteria’s ability to evade immune clearance. For the antibody response, high titers of OMV-, LPS- and CPS-specific antibodies were found in the sera with IgG1/IgG2a rates indicating a Th2-type antibody response in all cases. The study suggested a possible cross-protection between different strains due to a conserved LPS profile, despite the different virulence phenotypes in mice [26].

That study also included a passive immunization experiment using 300 μl of pooled sera from immunized or control mice. One hour after sera transfer, mice were i.p. challenge with 50 LD₅₀ of B. pseudomallei K96243, and survival was monitored for 14 days. In this case, 80% of passively immunized mice survived after 2 weeks, while all of the control group died 1–3 days post-infection. OMV-immune sera were also incubated with B. pseudomallei K96243 to determine if it retained antibacterial activity in vitro, and results indicated that the numbers of bacteria were reduced after 4 h of exposure to OMV-immune serum [26].

Finally, both safety and immunogenicity profiles of OMV-vaccines were evaluated in rhesus macaques as NHP animal model [7]. For that, two males were s.c. immunized at days 0, 28, and 56 with escalating doses of OMV vaccine (25, 50, and 100 μg, respectively) in combination with 400 μg of CpG ODN adjuvant. Neither changes in liver or kidney function nor erythema, swelling, or necrosis in the injection site were observed after OMV-immunization. Further, results showed high titers of OMV-, LPS-, and CPS-specific IgG in serum compared to control animals.

2. Conclusion

The scope of this review is restricted to highlight some B. pseudomallei vaccines that are currently funded by US agencies. This important financial investment has resulted in significant progress for the development and advancement of these vaccine candidates. The development and testing spectrum of these vaccines has been performed in the past 6–9 years, and in that period, advancement in the understanding about the major immune correlates of protection has been well established. The experimental data generated in the murine models of infection indicate that antibody production is critical to provide protection, and limited sterilizing immunity has been achieved in some vaccination schemes [11,23,32]. Further, evidence indicates that the selection of antigen combinations in the vaccine formulations is important to generate an effective humoral response, considering IgG2a is a critical antibody for protection in experimental models of melioidosis infection. However, production of IgG1 also seems to be important.

In the case of cellular immune responses, these are expected to be required for full protection due to the nature of this intracellular bacterial infection. However, the experimental data have not been able to produce a clear picture about the relevance of these responses. For CD8⁺ T cell responses, they appear to play a small role in protection against murine melioidosis disease [11,23]. This is in contrast to what is observed in humans, where CD8⁺ responses are linked to increased survival [48]. Furthermore, the evidence in murine melioidosis models and human infections strongly suggest that CD4⁺ T cell responses are required for full protection [23,48]. Finally, data strongly suggest that the production of IFN-γ and IL-17A correlate with B. pseudomallei killing (mouse and human) and with full protection.

Overall, the exciting progress with these vaccines indicates that with further optimization and experimentation, a more complete picture of the correlates of protection can emerge and be used as an informative platform to advance the vaccine candidates to the next stage of development.

3. Expert opinion

This is an exciting time in the field of melioidosis vaccinology because a milestone was reached in the development of vaccines that offer different levels of effective protection. In 2015, the Steering Group on Melioidosis Vaccine Development advised that funding should be pursued to perform a head-to-head comparison of candidate vaccines by an independent entity with the goal of identifying a vaccine formulation that can be moved forward into the developmental pipeline [22]. In response to this request, the Defense Threat Reduction Agency (DTRA) recently funded a project to the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) to perform a head-to-head comparison of all the DTRA-funded vaccines described in this review (OMVs, glycoconjugate vaccine, and live-attenuated vaccines). The study is planned to occur in the beginning of 2020, and it will include a prime and boost immunization protocol using C57BL/6 mice followed by an aerosol challenge with B. pseudomallei K96243. The expectation of the study is to provide solid evidence for antibody-mediated and cytokine responses elicited by the vaccines that confer protection and ideally, sterilizing immunity. As recommended by SGMVD, the head-to-head comparison of such vaccines in this standardized mouse model of melioidosis infection will provide key evidence for further studies in other pre-clinical platforms, including testing in a non-human primate model of melioidosis infection [22]. It is important that at this point, a careful selection of the optimal candidate vaccine(s) is done for future clinical studies using the proposed side-by-side gating approach.

Another important area of research should focus on the evaluation of vaccines in diabetic patients, a growing population worldwide, which represents a population susceptible to melioidosis disease. Vaccines that have been tested in different mouse models of infection and that have led in the head-to-head comparisons could be further evaluated in a diabetic mouse model of melioidosis prior to being evaluated in an NHP model of infection; however, this step is not necessarily essential for further advancement. Finally, investigators in the melioidosis field are cognizant that the selected vaccine might not provide sterilizing immunity and therefore, new lines of investigation should focus on combinatorial therapy, where vaccination would allow antibiotic treatment to be used more effectively because the window of time for antibiotic/antimicrobial intervention could be extended with vaccine use.

Through this review, we have presented the most recent, state-of-the-art research in the field of melioidosis vaccinology, which is funded by US agencies, and which is the pre-amble for developing a robust set of criteria for advancing one or several of these candidates from the mouse model stage to the NHP model and eventually to phase I through III clinical trials. As recommended by different experts, many of them part of SGMVD, the initial safety, and immunogenicity phase I and phase II human clinical studies should be performed in healthy volunteers, and the best candidate(s) should be examined in further clinical studies that must include diabetic volunteers. The impact of vaccination in endemic areas has been recently analyzed, and the data confirmed that there is a viable melioidosis vaccine market, but developers need to consider cost-effective vaccine strategies if there is to be successful implementation in most countries/territories where there is local transmission [49].

Article highlights.

• B. pseudomallei causes melioidosis, an infectious disease of subtropical and tropical areas of the world affecting both humans and animals.

• Melioidosis is known as the ‘great mimicker’ due to highly diverse disease manifestations, making diagnosis more difficult and delaying treatment.

• Leading B. pseudomallei vaccine candidates include OMVs, live-attenuated, glycoconjugate and nanoparticle vaccines.

• Head-to-head comparison of several of these vaccines in a standardized mouse model of melioidosis infection will provide key information about antibodies and cellular immune responses that correlate with protection ahead of pre-clinical studies.

• The safety and cost-effectiveness of vaccines against melioidosis need to be evaluated for healthy populations in endemic areas as well at-risk populations elsewhere.