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Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei . In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG 1 , IgG 2c , and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species., Competing Interests: The authors declare no conflict of interest.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

Melioidosis is a fatal tropical disease caused by the environmental Gram-negative bacterium, Burkholderia pseudomallei. This bacterium is intrinsically resistant to several antibiotics and treatment of melioidosis requires prolonged antibiotic administration. To date, there are no vaccines available for melioidosis. Previous studies have shown that humoral immunity is critical for surviving melioidosis and that O-polysaccharide (OPS) and hemolysin coregulated protein 1 (Hcp1) are important protective antigens in animal models of melioidosis. Our previous studies revealed that melioidosis patients had high levels of OPS- and Hcp1-specific antibodies and that IgG against OPS (IgG-OPS) and Hcp1 (IgG-Hcp1) were associated with patient survival. In this study, we characterized the potential function(s) of IgG-OPS and IgG-Hcp1 from melioidosis patients. IgG-OPS and IgG-Hcp1 were purified from pooled serum obtained from melioidosis patients using immuno-affinity chromatography. Antibody-dependent cellular phagocytosis assays were performed with pooled serum from melioidosis patients and compared with serum obtained from healthy controls. Serum from melioidosis patients significantly enhanced B. pseudomallei uptake into the human monocytic cell line THP-1 compared with pooled serum from healthy donors. Enhanced opsonization was observed with IgG-OPS and IgG-Hcp1 in a dose-dependent manner. Antibody-dependent complement deposition assays were performed with IgG-OPS and IgG-Hcp1 using flow cytometry and showed that there was enhanced C3b deposition on the surface of B. pseudomallei treated with IgG-OPS but to a lesser degree with IgG-Hcp1. This study provides insight into the function of IgG-OPS and IgG-Hcp1 in human melioidosis and supports that OPS and Hcp1 are potential vaccine antigens for immunization against melioidosis.

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. mallei ΔtonB Δhcp1 (CLH001). Thus, we applied this successful approach to the development of a similar vaccine against melioidosis by constructing the B. pseudomallei ΔtonB 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. pseudomallei IMPORTANCE 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., (Copyright © 2019 Khakhum et al.)

Burkholderia pseudomallei, the causative agent of melioidosis, is able to survive extreme environments and utilizes various virulence factors for survival and pathogenicity. To compete and survive within these different ecological niches, B. pseudomallei has evolved specialized pathways, including the Type VI secretion systems (T6SSs), that have a role in pathogenesis as well as interbacterial interactions. We examined the expression profile of B. pseudomallei T6SS six gene clusters during infection of U937 macrophage cells. T6SS-5 was robustly transcribed while the other five clusters were not significantly regulated proposing the utility of T6SS-5 as a potential biomarker of exposure to B. pseudomallei. Transcription of T6SS regulators VirAG and BprB was also not significant during infection when compared to bacteria grown in culture. Guided by these findings, three highly expressed T6SS genes, tssJ-4, hcp1 and tssE-5, were expressed as recombinant proteins and screened against melioidosis patient sera by western analysis and ELISA. Only Hcp1 was reactive by both types of analysis. The recombinant Hcp1 protein was further evaluated against a cohort of melioidosis patients (n = 32) and non-melioidosis individuals (n = 20) sera and the data clearly indicates a higher sensitivity (93.7%) and specificity (100%) for Hcp1 compared to bacterial lysate. The detection of anti-Hcp1 antibodies in patients' sera indicating the presence of B. pseudomallei highlights the potential of Hcp1 to be further developed as a serodiagnostic marker for melioidosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective To analyze the mechanism that Hcp1 protein in type Ⅵ secretion system of Burkholderia pseudomallei (B. pseudomallei) mediates the formation of multinucleated giant cells (MNGCs) when host cells are infected by the bacterium. Methods The mutant strain (BPC006 Δhcp1) and complementation strain (BPC006 Δhcp1:: hcp1) were constructed by homologous recombination and plasmid complement technology, respectively. After RAW264.7 cells were infected with B. pseudomallei, the localization of Hcp1 in host cells was analyzed by immunofluorescence staining. The localization was further verified by cytoplasmic-membrane isolation in 293T cells after transfecting pCDNA4.1-Hcp1. The biological significance and effect of Hcp1 were explored by the anti-Hcp1 polyclonal antibody blocking and the formation of MNGC was detected by Giemsa staining. Results Western blotting showed that BPC006 Δhcp1 could not express Hcp1, while BPC006 Δhcp1:: hcp1 restored Hcp1 expression. The above results proved that the mutant and complement strains were successfully constructed. Both cellular immunofluorescence co-localization and cytoplasmic-membrane isolation experiments showed that Hcp1 localized to host cell membranes. Last but not least, compared with the control group, anti-Hcp1 polyclonal antibodies inhibited the formation of MNGC (P < 0.01). Conclusion Hcp1 protein in type Ⅵ secretion system of B. pseudomallei is able to translocate to the RAW264.7 cell membranes and plays an important role in the formation of MNGCs.

Melioidosis, a severe tropical illness caused by Burkholderia pseudomallei, poses significant treatment challenges due to limited therapeutic options and the absence of effective vaccines. The pathogen's intrinsic resistance to numerous antibiotics and propensity to induce sepsis during acute infections further complicate management strategies. Thus, exploring alternative methods for prevention and treatment is crucial. Monoclonal antibodies (mAbs) have emerged as a promising strategy for the prevention and treatment of infectious diseases. This study focused on generating three mAbs (13F1, 14G11, and 15D9) targeting hemolysin-coregulated protein 1 (Hcp1), a protein involved in the type VI secretion system cluster 1 (T6SS1) of B. pseudomallei. Notably, pretreatment with 13F1 mAb significantly reduced the intracellular survival of B. pseudomallei and inhibited the formation of macrophage-derived multinucleated giant cells (MNGCs). This protective effect was also observed in vivo. We identified a sequence of amino acids (Asp95-Leu114) within Hcp1 as the likely binding site for 13F1 mAb. In summary, our findings reveal that 13F1 mAb counteracts infection by targeting Hcp1, offering potential new targets and insights for melioidosis prevention. [ABSTRACT FROM AUTHOR]

Melioidosis, a severe tropical illness caused by Burkholderia pseudomallei, poses significant treatment challenges due to limited therapeutic options and the absence of effective vaccines. The pathogen’s intrinsic resistance to numerous antibiotics and propensity to induce sepsis during acute infections further complicate management strategies. Thus, exploring alternative methods for prevention and treatment is crucial. Monoclonal antibodies (mAbs) have emerged as a promising strategy for the prevention and treatment of infectious diseases. This study focused on generating three mAbs (13F1, 14G11, and 15D9) targeting hemolysin-coregulated protein 1 (Hcp1), a protein involved in the type VI secretion system cluster 1 (T6SS1) of B. pseudomallei. Notably, pretreatment with 13F1 mAb significantly reduced the intracellular survival of B. pseudomallei and inhibited the formation of macrophage-derived multinucleated giant cells (MNGCs). This protective effect was also observed in vivo. We identified a sequence of amino acids (Asp95-Leu114) within Hcp1 as the likely binding site for 13F1 mAb. In summary, our findings reveal that 13F1 mAb counteracts infection by targeting Hcp1, offering potential new targets and insights for melioidosis prevention.

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.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei , is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. mallei ΔtonB Δhcp1 (CLH001). Thus, we applied this successful approach to the development of a similar vaccine against melioidosis by constructing the B. pseudomallei ΔtonB 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. pseudomallei. IMPORTANCE 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.