Burkholderia pseudomallei is an infectious bacterium of clinical and biodefense concern, and is the causative agent of melioidosis. The mortality rate can reach up to 50% and affects 165,000 people per year; however, there is currently no vaccine available. In this study, we examine the antigen-specific immune response to a vaccine formulated with antigens derived from an outer membrane protein in B. pseudomallei, Bucl8. Here, we employed a number of bioinformatic tools to predict Bucl8-derived epitopes that are non-allergenic and non-toxic, but would elicit an immune response. From these data, we formulated a vaccine based on two extracellular components of Bucl8, the β-barrel loops and extended collagen and non-collagen domains. Outbred CD-1 mice were immunized with vaccine formulations—composed of recombinant proteins or conjugated synthetic peptides with adjuvant—to assess the antigen-specific immune responses in mouse sera and lymphoid organs. We found that mice vaccinated with either Bucl8-derived components generated a robust TH2-skewed antibody response when antigen was combined with the adjuvant AddaVax, while the TH1 response was limited. Mice immunized with synthetic loop peptides had a stronger, more consistent antibody response than recombinant protein antigens, based on higher IgG titers and recognition of bacteria. We then compared peptide-based vaccines in an established C57BL/6 inbred mouse model and observed a similar TH2-skewed response. The resulting formulations will be applied in future studies examining the protection of Bucl8-derived vaccines.

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

Increasing antimicrobial resistance due to misuse and overuse of antimicrobials, as well as a lack of new and innovative antibiotics in development has become an alarming global threat. Preventative therapeutics, like vaccines, are combative measures that aim to stop infections at the source, thereby decreasing the overall use of antibiotics. Infections due to Gram-negative pathogens pose a significant treatment challenge because of substantial multidrug resistance that is acquired and spread throughout the bacterial population. Burkholderia spp. are Gram-negative intrinsically resistant bacteria that are responsible for environmental and nosocomial infections. The Burkholderia cepacia complex are respiratory pathogens that primarily infect immunocompromised and cystic fibrosis patients, and are acquired through contaminated products and equipment, or via patient-to-patient transmission. The Burkholderia pseudomallei complex causes percutaneous wound, cardiovascular, and respiratory infections. Transmission occurs through direct exposure to contaminated water, water-vapors, or soil, leading to the human disease melioidosis, or the equine disease glanders. Currently there is no licensed vaccine against any Burkholderia pathogen. This review will discuss Burkholderia vaccine candidates derived from outer membrane proteins, OmpA, OmpW, Omp85, and Bucl8, encompassing their structures, conservation, and vaccine formulation.

Burkholderia pseudomallei is a gram-negative bacterium that is the etiological agent of the tropical disease melioidosis. Currently, there is no licensed vaccine for melioidosis, but numerous candidates are being tested for protective efficacy and characterization of the elicited immune response. Our lab has previously reported the immunogenicity of a Bucl8-protein-based peptide antigen, designated L1-CRM197 (Cross-reacting material 197). When given subcutaneously, this vaccine formulation promoted a strong Th2 (IgG1) antibody response, however immunization did not protect from death. In this study, we hypothesized that an intranasally administered L1-CRM197 vaccine would induce protective mucosal immunity. To evaluate vaccine efficacy, we developed a surrogate Burkholderia infection model that employs outbred CD-1 mice which imitates the immunogenetic diversity of humans. Mice were immunized with either L1-CRM197 adjuvanted with fluorinated cyclic diguanosine monophosphate (FCDG) or with FCDG-only control. These mice were then challenged intranasally with an infectious dose of a luminescent strain of B. thailandensis E264 two weeks post-immunization, and correlates of protection were assessed in euthanized mice on days 1, 2, 3, and 7 post-infection. Overall, intranasal vaccination, compared to subcutaneous administration, induced a stronger Th1 (IgG2a/2b) to Th2 (IgG1) antibody response and promoted anti-L1 nasal, pulmonary, and systemic IgA. Additionally, sera IgG from L1-CRM197-vaccinated mice recognized whole-cell B. thailandensis and B. pseudomallei, a select agent exempt strain Bp82. Vaccination ameliorated disease indicators, including luminescent signal and bacterial cell counts, weight and temperature loss, and organ weight, which negatively correlated with IgG2a antibody levels and mucosa-stimulating cytokines IL-13 and IL-9. L1-CRM197-vaccinated mice also had earlier resolution of inflammatory and tissue-damaging cytokines compared to the FCDG-only controls. These results suggest a balanced humoral and cell-mediated response, along with mucosa-based immunity are beneficial for protection. Future efforts should further assess mucosal cellular and humoral mechanisms of protection and test such protection, using aerosolized B. pseudomallei select agent strain(s).

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