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Bacterial gastrointestinal diseases

Development of technology for blood-clean treatment of Vibrio vulnificus sepsis-induced antigens

V. vulnificus causes illness ranging from gastrointestinal diseases to ‘primary sepsis’ and necrotizing fasciitis. The case-fatality rate reportedly exceeds 50% in primary sepsis. Bio-spleen technology using nanobeads has been studied recently, and we are developing this technology as a treatment for sepsis. This study aims to remove from blood the three substances that cause symptoms as follows: (a) removal of RTXA1 secreted by V. vulnificus that causes necrosis of blood cells; (b) removal of V. vulnificus using bacterial and adherent cellular substances; and (c) treatment of the symptoms of sepsis by inhibition of iron transport essential for bacteria survival. To remove RTXA1 using a monoclonal antibody, the C-terminal region of about 100 KDa was recombined and purified. Five purified monoclonal antibodies were produced by cell fusion after immunization with purified proteins. The epitope site of each antibody is currently being screened. The binding efficiency of V vulnificus was evaluated by coating magnetic beads (MB) with fibronectin, laminin, and collagen I and IV. The efficiency of fibronectin was 100-fold higher than that of the control group and approximately 10-fold higher than that of collagen IV. For the iron transport inhibition, the receptor of VuuA (VuuA) was purified as a recombinant protein and coated on Dynabead M-270 epoxy. The in vitro growth rate was measured in lysogeny broth (LB) and chelated LB to confirm inhibition of bacterial growth by functionalized MB. The growth rate in the VuuA-MB-treated group was significantly lower than that of the control group; in particular, growth was completely inhibited in chelated LB, similar to blood conditions (Figure 9).

Figure 9. Growth inhibition of CMCP6 cells by VuuA-MB and an adherence assay with extracellular matrix.
Figure 1. Growth inhibition of CMCP6 cells by VuuA-MB and an adherence assay with extracellular matrix.


Tuberculosis

Evaluation of the protective efficacy of a tuberculosis vaccine candidate originating from nontuberculosis mycobacteria (NTM)

Incomplete protection efficacy of BCG is a driving force for the development of new tuberculosis (TB) vaccines. The capacity of the SNUMI-9 vaccine candidate, based on NTM, has been evaluated by challenge with Mycobacterium tuberculosis. We examined the therapeutic effect of the SNUMI-9 vaccine against TB infection in C57BL/6 mice. The mice were infected with 100 colony-forming units (CFUs) of M. tuberculosis by inhalation exposure. Starting at the fourth week post-infection until sixth week, mice were administered isoniazid (INH) chemotherapy and two doses of SNUMI-9 were administered for two weeks at the same time. The CFUs were counted using the 7H10 agar plating method and cytokine- produced purified protein derivative stimulation on mouse splenocytes was carried out by fluorescence-activated cell sorting (FACS) analysis. We used another virulence parameter, lung histopathology, to assess the therapeutic effect of SNUMI-9. Subcutaneous vaccination of live SNUMI-9 reduced viable bacterial counts in the lungs of mice after vaccination and INH treatment at a level similar to that of the BCG vaccine. Nine weeks after vaccination, live SNUMI-9-vaccinated mice had similar granulomatous inflammation as that of BCG-vaccinated mice. In contrast, PBS and heat-killed SNUMI-9-vaccinated mice progressed more quickly. Interferon-gamma (IFN-γ) induction was higher in live and heat-killed SNUMI-9 mice than those in BCG and PBS controls at three weeks after vaccination, but mouse serum IFN-γ levels did not differ. In conclusion, BCG and live SNUMI-9 showed a similar therapeutic effect in C57BL/6 mice, but SNUMI-9 induced greater CD4 T cell proliferation compared to that of BCG after short-term vaccine and chemotherapy treatment. Therefore, the live SNUMI-9 strain could be a candidate for inclusion in a therapeutic vaccine against TB.


Evaluation of Mycobacterium tuberculosis-specific antigen peptides to develop a diagnosis kit for latent TB infection (LTBI) detection

New biomarkers derived from M. tuberculosis have been investigated for the accurate diagnosis of TB. Multifaceted approaches are required to identify diagnostic antigens related to LTBI. Extensive analysis of the antigens and epitopes of M. tuberculosis K strain coupled with bioinformatics with reverse vaccinology predicted 10 novel antigen candidates inducing IFN-γ release for LTBI diagnosis. We also considered four proteins associated with active or latent TB infection. To validate the specificity and conservation, the genes encoding 14 proteins (K1 to K14) were analysed by gene amplification and sequencing for the vaccine strain, M. bovis BCG-pasteur 1173P2, 29 non-tuberculoid mycobacterial strains, and 134 clinical isolates of M. tuberculosis. All clinical isolates were selected for representativeness, considering drug resistance, region, and molecular type. None of the genes encoding the 14 proteins was detected in any NTM strains. Among 14 candidates, only the four genes encoding K3, K4, K6, and K12 were M. tuberculosis-specific not detected in the BCG strain. M. tuberculosis isolates with various molecular types harboured both K3 and K6 genes. The K4 and K12 genes were found only Beijing-type strains highly prevalent in Korea. These results demonstrate the potential of K3, K4, K6, and K12 as new diagnostic markers. We have been collecting blood samples from healthy controls as well as latent and active TB patients. From sera and peripheral blood mononuclear cell samples, K3, K4, K6, and K12 will be tested as diagnostic antigens for the detection of TB or discrimination between latent and active TB.



Zoonosis

Genetic and immunopathologic characterizations of a clinical isolate from an anaplasmosis case in South Korea

The obligate intracellular bacterium Anaplasma phagocytophilum causes human anaplasmosis. The bacterium is a tick-borne pathogen transmitted by Ixodes spp. ticks that infect domestic mammals and humans. In South Korea, 18 cases in 2015, 19 cases in 2016 and 91 cases in 2017 of human anaplasmosis have been reported annually since 2015, but very little is known about how A. phagocytophilum infects and spreads. In a previous study, we isolated A. phagocytophilum from a patient in South Korea. We investigated the structure of A. phagocytophilum by electron microscopy and performed a sequence analysis of major proteins.
In this study, A. phagocytophilum morulae were observed in HL-60 promyelocytic leukaemia cells by transmission electron microscopy (Figure 10). The msp2/p44 sequence of the isolate was dissimilar to those of A. phagocytophilum strains isolated in the United States and Sweden (Figure 11). We are currently undertaking further studies to elucidate the complete gene sequences for Korean A. phagocytophilum isolates.

Figure 10. Ultrastructure of Anaplasma phagocytophilum isolate by transmission electron microscopy. The replicating bacteria had nearly filled the morulae in HL-60 cells.
Figure 2. Ultrastructure of Anaplasma phagocytophilum isolate by transmission electron microscopy. The replicating bacteria had nearly filled the morulae in HL-60 cells.


Figure 11. Comparison of Anaplasma phagocytophilum msp2/p44 sequences. The arrows indicate sequence differences among A. phagocytophilum isolates.

Figure 3. Comparison of Anaplasma phagocytophilum msp2/p44 sequences. The arrows indicate sequence differences among A. phagocytophilum isolates.

Characteristics of Borrelia sp. and improved laboratory diagnosis

This study analysed the characteristics of a Korean Borrelia isolate and emphasized the importance of an improved serological laboratory test and the standardization of clinical laboratory co-operation for accurate diagnosis of Lyme disease (LD). The culture protocol for the standard strain, Borrelia afzelii S13, was established in our laboratory by optimizing various conditions (Figure 12). We cultured Borrelia bacteria isolates from 300 suspected samples and detected the target gene in two cultured samples (Figure 13).



Figure 12. Borrelia afzelii two (A) and five days (B) post-infection (Leica microsystem, DE/DM-250 phase-contrast microscope, Leica MC 170 HD camera (200x)).
Figure 4. Borrelia afzelii two (A) and five days (B) post-infection (Leica microsystem, DE/DM-250 phase-contrast microscope, Leica MC 170 HD camera (200x)).


Figure 13. Amplification of flaB gene using nested PCR in clinical samples.
Figure 5. Amplification of flaB gene using nested PCR in clinical samples.


 The seroprevalence of LD among national park workers considered to be at high risk of exposure to Borrelia species was investigated to compare two serological assays: indirect immunofluorescence antibody assays (IFA) and enzyme-linked immunosorbent assays (ELISA). A total of 763 sera samples were examined to detect immunoglobulin IgG or IgM antibodies to Borrelia species by two-tier serologic tests according to a laboratory diagnostic guideline for LD. Additionally, demographic data were obtained by a questionnaire survey. A total of 60 IFA positive cases (7.9%) and 243 ELISA positive cases (32.1%) were detected by screening assays. In western blot assays, only 3 and 5 cases were identified respectively, and the specificity of the ELISA assay should be considered. The results indicates that an improvement of the primary diagnostic method is required (Tables 1 and 2).
Although these results may be limited to certain groups, this study is important for determining the seroprevalence of populations at high risk of contracting LD.
Table 2. Comparison of IFA and ELISA results of seroreactive specimens from national park workers.
Table 1. Results of two screening assays of national park service workers for the serodiagnosis of LD based on IgG and IgM (A and B).


Table 2. Comparison of IFA and ELISA results of seroreactive specimens from national park workers.
Table 2. Comparison of IFA and ELISA results of seroreactive specimens from national park workers.

a Positive by IFA and negative by ELISA; b Positive by ELISA, negative by IFA;
two is duplicate in a total of 60 IFA positive cases and eleven is duplicate in 243 ELISA positive cases

* IFA or ELISA was performed for screening tests, with seropositivity defined as an IgG titre of ≥1:256 or IgM titre of ≥1:16 in IFA or an optical density ratio measured at 450 nm of reacting IgG or IgM with ≥1.1 in ELISA. Only seropositive samples with scores ≥7 points in western blot (WB) assay were confirmed as seroprevalent.

Advancement and evaluation of an ELISA kit for the diagnosis of scrub typhus

We investigated the 56-kDa type-specific antigen (TSA56) of the Karp, Kato, Gilliam, and Boryong Orentia tsutsugamushi strains as potential antigens for a high-throughput ELISA-based assay. The TSA56 genes were modified and optimized for an E. coli expression system. Synthetic TSA56 genes were expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Using sera from patients with confirmed tsutsugamushi disease, we tested the serological responses to mixtures of TSA56 proteins according to their mixing ratios. Additionally, various ELISA conditions such as antigen concentration and sample dilution buffer type were assessed. The reactivities of the mixtures of TSA56 proteins differed depending on their mixing ratios. ELISA using mixtures of these recombinant TSA56 proteins and 372 human serum samples demonstrated reliable sensitivity (90.11%) and specificity (91.58%) (Figure 14). For further application, verification with a large number of samples and validations of regional cut-off values for diagnostic accuracy should be considered.




Figure 14. Profiles of indirect ELISA with TSA56 mixtures from O. tsutsugamushi Karp, Kato, Gilliam, and Boryong strains. Sera from scrub typhus patients were confirmed by PCR and IFA with anti-IgG antibody. Sera were titrated from 1:16 to 1:2048. Negative sera were obtained from healthy individuals and patients with other acute febrile illness (Q-fever, Lyme disease, and Bartonellosis) which do not react with O. tsutsugamushi.
Figure 6. Profiles of indirect ELISA with TSA56 mixtures from O. tsutsugamushi Karp, Kato, Gilliam, and Boryong strains. Sera from scrub typhus patients were confirmed by PCR and IFA with anti-IgG antibody. Sera were titrated from 1:16 to 1:2048. Negative sera were obtained from healthy individuals and patients with other acute febrile illness (Q-fever, Lyme disease, and Bartonellosis) which do not react with O. tsutsugamushi.

Development of a molecular diagnostic tool for scrub typhus

Enhanced molecular diagnostic tools for detecting and differentiating O. tsutsugamushi serotypes based upon PCR were developed. The specific primer sets were devised to target the 56-kDa outer membrane protein of the Kato, Gilliam, Karp, and Boryong strains. Each type of genomic DNA was isolated and purified from culture supernatants of L929 cells infected with each O. tsutsugamushi strain (Figure 15).



Figure 15. Representative IFA image of O. tsutsugamushi Boryong. Reactions of tsutsugamushi-infected patients’ serum with O. tsutsugamushi Boryong with fluorescein isothiocyanate (FITC)-labelled goat, IgG anti-human antibody (40x magnification).
Figure 7. Representative IFA image of O. tsutsugamushi Boryong. Reactions of tsutsugamushi-infected patients’ serum with O. tsutsugamushi Boryong with fluorescein isothiocyanate (FITC)-labelled goat, IgG anti-human antibody (40x magnification).

The designed primer sets were tested for specificity (Figure 16). All primer sets showed proper specificity and a universal detection primer showed similar results. This is the first step in the development of enhanced methods for the diagnosis of O. tsutsugamushi, and these results might be the scientific scaffolding to develop effective diagnostic tools based on molecular biological techniques.

Figure 16. Detection of rickettsial DNA using type-specific primers by conventional PCRs. Agarose gel electrophoresis of rickettsial DNA amplified by PCRs from serotype-specific and universal primers.
Figure 8. Detection of rickettsial DNA using type-specific primers by conventional PCRs. Agarose gel electrophoresis of rickettsial DNA amplified by PCRs from serotype-specific and universal primers.
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