Full-length 16S rDNA sequencing based on Oxford Nanopore Technologies revealed the association between gut-pharyngeal microbiota and tuberculosis in cynomolgus macaques.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (-) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future.

Advancing tuberculosis diagnosis and management in cynomolgus macaques using Xpert MTB/RIF ultra assay.

The detection and management of Mycobacterium tuberculosis complex (MTBC) infection, the causative agent of tuberculosis (TB), in macaques, including cynomolgus macaques (Macaca fascicularis), are of significant concern in research and regions where macaques coexist with humans or other animals. This study explored the utility of the Xpert MTB/RIF Ultra assay, a widely adopted molecular diagnostic tool to diagnose tuberculosis (TB) in humans, to detect DNA from the Mycobacterium tuberculosis complex in clinical samples obtained from cynomolgus macaques. This investigation involved a comprehensive comparative analysis, integrating established conventional diagnostic methodologies, assessing oropharyngeal-tracheal wash (PW) and buccal swab (BS) specimen types, and follow-up assessments at 3-month, 6-month, and 12-month intervals. Our results demonstrated that the Xpert MTB/RIF Ultra assay was able to detect MTBC in 12 of 316 clinical samples obtained from cynomolgus macaques, presenting a potential advantage over bacterial culture and chest radiographs. The Xpert MTB/RIF Ultra assay exhibited exceptional sensitivity (100%) at the animal level, successfully detecting all macaques positive for M. tuberculosis as confirmed by traditional culture methods. The use of PW samples revealed that 5 positive samples from 99 (5.1%) were recommended for testing, compared to 0 samples from 99 buccal swab (BS) samples (0.0%). In particular, the definitive diagnosis of TB was confirmed in three deceased macaques by MTB culture, which detected the presence of the bacterium in tissue autopsy. Our findings demonstrate that the implementation of the Xpert MTB/RIF Ultra assay, along with prompt isolation measures, effectively reduced active TB cases among cynomolgus macaques over a 12-month period. These findings highlight the advance of the Xpert MTB/RIF Ultra assay in TB diagnosis and its crucial role in preventing potential outbreaks in cynomolgus macaques. With its rapidity, high sensitivity, and specificity, the Xpert MTB/RIF Ultra assay can be highly suitable for use in reference laboratories to confirm TB disease and effectively interrupt TB transmission.

The Adjuvant Activity of BCG Cell Wall Cytoskeleton on a Dengue Virus-2 Subunit Vaccine.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has made it difficult to achieve balanced protection against all four serotypes of the dengue virus (DENV). To overcome this problem, non-replicative vaccines have come into focus, as their immunogenicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composed of UV-inactivated dengue virus-2 (UV-DENV-2) and DENV-2 NS1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by BCG-CWS significantly augmenting antibody-mediated complement-fixing activity, strongly stimulating the antigen-specific polyfunctional T cell responses, and activating mixed Th-1/Th-2 responses specific to DENV-2- and NS1-279 antigens. In conclusion, BCG-CWS potently adjuvanted the inactivated DENV-2 and DENV subunit immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

Non-invasive specimen collections for Mycobacterium tuberculosis detection in free-ranging long-tailed macaques (Macaca fascicularis).

Surveillance of infectious diseases in free-ranging or wild animals has been widely conducted in many habitat-range countries after the COVID-19 episode. Thailand is located in the center of the distribution range of long-tailed macaques (Macaca fascicularis; Mf) where the animals have both frequent human contact and a high prevalence of human tuberculosis. For the large-scale detection of Mycobacterium tuberculosis complex (MTBC) using IS6110-nested PCR in free-ranging Mf, non-invasive sampling was developed using oral (via rope bait) and fecal (direct swabs of fresh feces) specimen collection. Firstly, the MTBC-IS6110-nested PCR was validated in non-invasively collected specimens, in terms of its specificity and sensitivity, and then compared with those of the invasively collected oral and rectal swabs in 24 captive MTBC-suspected Mf. After validation, these methods were applied to survey for the prevalence of shed MTBC (MTBCS) in four previously reported MTBC-infected populations. A total of 173 baited rope specimens and 204 freshly defecated excretions were collected. The limit of detection of the IS6110-nested PCR technique was 10 fg/µL and the 181-bp PCR amplicon showed 100% sequence similarity with the MTB H37Rv genome sequence. Comparing the MTBCS detection between the invasive and non-invasive collected specimens in captive suspected Mf revealed a significant correlation between the two types of oral specimens (oral swabs and baited ropes; n = 24, r2 = 1, p-value < 0.001), but fresh fecal swabs showed higher MTBCS frequencies than the rectal swabs. Moreover, the proportion of MTBCS-positive free-ranging Mf were significantly higher in the fresh fecal swabs (8.82%; 95% CI; 4.9-12.7%) than in the baited ropes (5.20%; 95% CI; 1.9-8.5%). This result indicates that oral sampling via baited ropes and fecal sampling via defecated excretion swabs can serve as ancillary specimens for MTBCS detection in free-ranging non-human primates.

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus).

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Alteration of gut microbiota in wild-borne long-tailed macaques after 1-year being housed in hygienic captivity.

The wild-born long-tailed macaques (Macaca fascicularis) were recently recruited and used as breeders for the National Primate Research Center of Thailand, Chulalongkorn University (NPRCT-CU), and changes in their in-depth gut microbiota profiles were investigated. The Oxford Nanopore Technology (ONT) was used to explore full-length 16S rDNA sequences of gut microbiota in animals once captured in their natural habitat and 1-year following translocation and housing in a hygienic environment at NPRCT-CU. Our findings show that the gut microbiota of macaques after 1 year of hygienic housing and programmed diets feeding was altered and reshaped. The prevalent gut bacteria such as Prevotella copri and Faecalibacterium prausnitzii were enriched after translocation, causing the lower alpha diversity. The correlation analysis revealed that Prevotella copri, Phascolarctobacterium succinatutens, and Prevotella stercorea, showed a positive correlation with each other. Significantly enriched pathways in the macaques after translocation included biosynthesis of essential amino acids, fatty acids, polyamine and butanoate. The effects of microbiota change could help macaques to harvest the energy from programmed diets and adapt their gut metabolism. The novel probiotics and microbiota engineering approach could be further developed based on the current findings and should be helpful for captive animal health care management.

Peptide microarray-based identification of dormancy-associated Mycobacterium tuberculosis antigens inducing immune responses among latent tuberculosis infection individuals in Thailand.

Multi-stage tuberculosis (TB) vaccines composed of active- and dormancy-associated antigens are promising to trigger the immune protection against all TB stages. However, scientists are still in quest of the suitable vaccine candidates. In this study, we identified the potential targets for this vaccine in a high TB burden country, Thailand. Peptide microarray was applied to gauge IgA and IgG antibodies specific to 16,730 linear epitopes of 52 dormancy-associated Mycobacterium tuberculosis (M. tb) proteins in three study groups: active tuberculosis (ATB), latent tuberculosis infection (LTBI) and endemic healthy control (EHC). Preferential IgA recognition against epitopes of dormancy-associated proteins was identified in LTBI group. Validation of these findings revealed that LTBI subjects exhibited the greater levels of Rv2659c- and Rv1738-specific IgA than those of household contacts, but less than did ATB subjects. Frequencies of IFNγ-producing CD4+ and CD8+ T cells induced by proteins Rv2659c and Rv1738 were higher in LTBI than ATB individuals. The results indicated that LTBI group in a high TB burden country demonstrated cell-mediated immune response to proteins Rv2659c and Rv1738 stronger than those of ATB. These immune responses likely contribute to natural protection against dormant M. tb and might be potential targets for a multi-stage TB vaccine.

Prevalence of Mycobacterium tuberculosis Complex among Wild Rhesus Macaques and 2 Subspecies of Long-Tailed Macaques, Thailand, 2018-2022.

We identified tuberculosis in 1,836 macaques from 6 wild rhesus (Macaca mulatta), 23 common long-tailed (M. fascicularis fascicularis), and 6 Burmese long-tailed (M. fascicularis aurea) macaque populations in Thailand. We captured, anesthetized, and collected throat, buccal, and rectal swab specimens from the macaques. We screened swabs for Mycobacterium tuberculosis complex (MTBC) using insertion sequence 6110-specific nested PCR. We found higher MTBC prevalence at both population and individual levels among M. mulatta than M. fascicularis fascicularis macaques; all 3 M. fascicularis aurea macaque populations were positive for tuberculosis. We found that throat swab specimens provided the best sample medium for detecting MTBC. Our results showed no difference in MTBC prevalence between male and female animals, but a higher percentage of adults were infected than subadults and juveniles. Although we detected no association between frequency of human-macaque interaction and MTBC prevalence, bidirectional zoonotic transmission should be considered a possible public health concern.

Tuberculosis detection in nonhuman primates is enhanced by use of testing algorithms that include an interferon-γ release assay.

OBJECTIVE: To develop a testing algorithm that incorporates multiple assays to evaluate host cellular and humoral immunity and antigen detection concerning Mycobacterium tuberculosis complex (MTBC) infection in captive nonhuman primates. ANIMALS: Cohorts of captive-bred and wild-caught macaques from 5 different geographic regions. PROCEDURES: Macaques were tested for MTBC infection by use of a γ interferon tuberculosis (GIFT) assay, an interferon-γ release assay, and other assays. In the first 2 cohorts (n = 15 and 181), initial validation of the GIFT assay was performed by use of experimentally infected and unexposed control macaques. In the next 3 cohorts (n = 59, 42, and 11), results were obtained for opportunistically collected samples from macaques exposed during spontaneous outbreaks. RESULTS: Sensitivity and specificity of the GIFT assay in the control cohorts were 100% and 97%, respectively, and were variable but enhanced by incorporating results from multiple assays in spontaneous outbreaks. CLINICAL RELEVANCE: The detection and management of MTBC infection in captive nonhuman primate populations is an ongoing challenge, especially with animal imports and transfers. Despite standardized practices of initial quarantine with regular intradermal tuberculin skin testing, spontaneous outbreaks continue to be reported. Since infection encompasses a range of disease manifestations over time, a testing algorithm that incorporates multiple assays, such as the GIFT assay, to evaluate host cellular and humoral immunity in addition to agent detection is needed. Testing a combination of samples from controlled studies and spontaneous outbreaks of MTBC infection in nonhuman primates would advance the development and validation of a functional algorithm that incorporates promising tools such as the GIFT assay.

Circulating IgA/IgG memory B cells against Mycobacterium tuberculosis dormancy-associated antigens Rv2659c and Rv3128c in active and latent tuberculosis.

OBJECTIVE: To elucidate the antigenic potential of dormancy-associated antigens Rv2659c and Rv3128c of Mycobacterium tuberculosis by examining the persistence of specific IgG and IgA memory B cells (MBCs) among patients with active tuberculosis (TB), household contacts with latent tuberculosis (LTBI), and an endemic healthy control group. METHODS: Fresh peripheral blood mononuclear cells from the three study groups were used to enumerate the numbers of IgG and IgA MBCs specific to recombinant protein Rv2659c and Rv3128c by ELISpot assay. The composition of MBC subsets IgA+ and IgG + was analyzed by flow cytometry. RESULTS: The number of IgA MBCs specific to antigen Rv2659c was significantly higher in the LTBI group than the TB group. In contrast, no significant difference was found in IgA or IgG MBCs against antigen Rv3128c. The number of IgA+ MBCs was significantly higher than that of IgG+ MBCs in the classical MBC subset of the LTBI group. CONCLUSION: The results indicated that the dormancy-associated antigen Rv2659c induced an IgA MBCs response in individuals with latent TB, and IgA+ classical MBCs formed a major portion of the MBCs subset. This new knowledge will be beneficial for the development of novel TB vaccines and their control of latent TB.

Whole inactivated dengue virus-loaded trimethyl chitosan nanoparticle-based vaccine: immunogenic properties in ex vivo and in vivo models.

Dengue virus (DENV) is a mosquito-borne virus that poses an incomparable public health problem, particularly in tropical and subtropical areas. Vaccination remains the most rational measure for controlling DENV infection. In this study, an ultraviolet irradiation (UV)-inactivated DENV-2 carried by N,N,N-trimethyl chitosan nanoparticles (UV-inactivated DENV2 TMC NPs) was investigated as a potential non-replicating dengue vaccine candidate. Using a human ex vivo model, the human monocyte-derived dendritic cells (MoDCs), we showed that TMC served as both a vaccine vehicle and a potent adjuvant. TMC NPs not only efficiently enhanced UV-inactivated DENV2 internalization into MoDCs but also greatly increased the breadth of UV-inactivated DENV2 immunogenicity to drive the maturation of MoDCs. Moreover, UV-inactivated DENV2 TMC NPs were highly immunogenic in mice, inducing greater levels of antibodies (total IgG, IgG1, IgG2a and neutralizing antibodies) and T cells (activated CD4⁺ and CD8⁺ T cells) against DENV-2 compared to soluble DENV-2 immunogens. Notably, the neutralizing activity of sera from mice immunized with UV-inactivated DENV2 TMC NPs was significantly augmented in the presence of complement activation, leading to the strong elimination of both DENV-2 particles and infected cells. We further showed that the immunogenicity of an inactivated dengue-based vaccine was significantly improved in a concentration-dependent manner. These positive results warrant further investigations of this platform of vaccine delivery for tetravalent vaccines or monovalent vaccines in sequential immunizations.

Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nucleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum.

The loop-mediated isothermal amplification coupled with lateral flow dipstick (PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparumdhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities (10 to 87,634 parasites per microliter of blood). The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.

Detection of Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus) using an interferon gamma release assay in a captive elephant herd.

Tuberculosis is highly contagious disease that can be transmitted between humans and animals. Asian elephants (Elephas maximus) in captivity live in close contact with humans in many Asian countries. In this study, we developed an interferon gamma release assay (IGRA) for elephant TB detection using antigens from the MTB complex (MTBC) and nontuberculous mycobacteria (NTM) as stimulating antigens (PPD, ESAT6, CFP10) to elicit a cell-mediated immune response (CMIR). The developed assay was applied to an elephant herd of more than 60 animals in Thailand, and the results were compared with those obtained through serological detection. IGRA has sufficient sensitivity for detecting elephant interferon gamma (eIFNγ) from specific antigen-stimulated PBMCs. Among 60 animals tested, 20 samples (33.3%) showed negative results for both MTBC and NTM infection. Eighteen samples (30%) showed positive responses against PPD from M. bovis and/or ESAT6 and CFP10, indicating MTBC infection. In contrast, only 15.6% showed seropositivity in a commercial serological test kit for elephant TB. The discrepancies between serological and CMIR highlight that the two methods may detect different stages of elephant TB. Therefore, employing both tests may enable them to complement each other in correctly identifying elephants that have been exposed to MTBC.

Nanodelivery system enhances the immunogenicity of dengue-2 nonstructural protein 1, DENV-2 NS1.

Nonstructural protein 1 (NS1) of dengue virus (DENV) is currently recognized as a dengue vaccine candidate. Unfortunately, most of non-replicating immunogens typically stimulate unsatisfactory immune responses, thus, the additional adjuvant is required. In this study, C-terminal truncated DENV-2 NS1 loaded in N,N,N, trimethyl chitosan nanoparticles (NS11-279TMC NPs) was prepared through the ionic gelation method. The immunogenicity of NS11-279TMC NPs was investigated using human ex vivo as well as the murine model. Through a human ex vivo model, it was demonstrated in this study that not only can TMC particles effectively deliver NS11-279 protein into monocyte-derived dendritic cells (MoDCs), but also potently stimulate those cells, resulting in increased expression of maturation marker (CD83), costimulating molecules (CD80, CD86 and HLA-DR) and markedly secreted various types of innate immune cytokines/chemokines. Moreover, mice administered with NS11-279TMC NPs strongly elicited both antibody and T cell responses, produced higher levels of IgG, IgG1, IgG2a and potently activated CD8+ T cells, as compared to mice administered with soluble NS11-279. Importantly, we further demonstrated that anti-NS11-279 antibody induced by this platform of NS11-279 effectively eliminated DENV-2 infected cells through antibody dependent complement-mediated cytotoxicity. Significantly, anti-DENV2 NS11-279 antibody exerted cross-antiviral activity against DENV-1 and -4 but not against DENV-3 infected cells. These findings demonstrate that TMC exerts a desirable adjuvant for enhancing delivery and antigenicity of NS1 based dengue vaccine.

Prevalence of asymptomatic Plasmodium infections with sub-microscopic parasite densities in the northwestern border of Thailand: a potential threat to malaria elimination.

BACKGROUND: Asymptomatic infections with sub-microscopic Plasmodium serve as a silent reservoir of disease, critical to sustaining a low level of remanent malaria in the population. These infections must be effectively identified and targeted for elimination. The sensitivity of light microscopy, the traditional method used for diagnosing Plasmodium infections, is frequently insufficient for detecting asymptomatic infections due to the low density of parasitaemia. The objective of this study was to explore the current prevalence of asymptomatic sub-microscopic Plasmodium carriages to evaluate the parasite reservoir amongst residents from 7 hamlets in Tak Province in northwestern Thailand using a highly sensitive molecular method. METHODS: Malaria infection was screened in a real-world setting from 3650 finger-prick blood specimens collected in a mass cross-sectional survey using light microscopy and loop-mediated isothermal amplification (LAMP). LAMP results were later confirmed in a laboratory setting in Bangkok using nested PCR, restriction enzyme digestion and DNA sequencing. The association of malaria infection with demographic factors was explored. RESULTS: Parasite prevalence was 0.27% (10/3650) as determined by microscopy. Sub-microscopic infection prevalence was 2.33% (85/3650) by LAMP. Of these, 30.6% (26/85) were infected with Plasmodium falciparum, 52.9% (45/85) with Plasmodium vivax, 2.4% (2/85) with Plasmodium malariae, 4.7% (4/85) with mixed P. falciparum and P. vivax, and 9.4% (8/85) had parasite densities too low for species identification. Asymptomatic carriages (T < 37.5 °C) accounted for 95% (76/80) of all sub-microscopic cases with the highest prevalence occurring in the subjects 31-45 years of age (p ≤ 0.035). Participants working on plantations or as merchants had an increased infection risk. Evaluation by microscopy identified 10.53% (10/95) of all Plasmodium infected participants. CONCLUSION: Participants carrying asymptomatic Plasmodium infections with sub-microscopic parasite densities are considerable in this area. These findings provide the true disease burden and risk factors in this region. This information helps to direct policy makers towards better schemes and delivery of targeted interventions. Moreover, this is the first study to use LAMP in mass screening for sub-clinical and sub-microscopic infections in a field setting in Thailand. LAMP proves to be a sensitive and field-deployable assay suitable for national malaria control screening campaigns.

In Vitro Activities of Enantiopure and Racemic 1'-Acetoxychavicol Acetate against Clinical Isolates of Mycobacterium tuberculosis.

In the process of evaluating the effect of several plant extracts against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA), an extract of Thai herb Alpinia galanga rhizome and its major component, 1'-acetoxychavicol acetate (ACA), exhibited marked anti-tuberculosis activity. The minimal inhibition concentrations (MICs) of the S-enantiomer of ACA (S-ACA) against M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294 strains were 0.2 µg/mL and 0.7 µg/mL, respectively. More than 95% of 100 drug-sensitive and 50 drug-resistant mycobacterial clinical isolates were inhibited by extracted S-ACA at 1.0 µg/mL. All of the remaining isolates were inhibited at 2.0 µg/mL. In contrast to the S-enantiomer, synthetic racemic 1'-R,S-ACA (rac-ACA) showed MICs of 0.5 µg/mL and 2.7 µg/mL for M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294, respectively, suggesting that the anti-tuberculosis effect might be primarily due to the S-form. These observations were in line with the MICs of rac-ACA against 98% of 93 drug-resistant clinical isolates, which showed the effective inhibitory dose at 2.0 µg/mL. After exposure to 2.7 µg/mL of rac-ACA for at least 3 h, the tubercle bacilli were completely killed. These demonstrated that ACA had potent anti-TB activity.

Immunogenic Properties of a BCG Adjuvanted Chitosan Nanoparticle-Based Dengue Vaccine in Human Dendritic Cells.

Dengue viruses (DENVs) are among the most rapidly and efficiently spreading arboviruses. WHO recently estimated that about half of the world's population is now at risk for DENV infection. There is no specific treatment or vaccine available to treat or prevent DENV infections. Here, we report the development of a novel dengue nanovaccine (DNV) composed of UV-inactivated DENV-2 (UVI-DENV) and Mycobacterium bovis Bacillus Calmette-Guerin cell wall components (BCG-CWCs) loaded into chitosan nanoparticles (CS-NPs). CS-NPs were prepared by an emulsion polymerization method prior to loading of the BCG-CWCs and UVI-DENV components. Using a scanning electron microscope and a zetasizer, DNV was determined to be of spherical shape with a diameter of 372.0 ± 11.2 nm in average and cationic surface properties. The loading efficacies of BCG-CWCs and UVI-DENV into the CS-NPs and BCG-CS-NPs were up to 97.2 and 98.4%, respectively. THP-1 cellular uptake of UVI-DENV present in the DNV was higher than soluble UVI-DENV alone. DNV stimulation of immature dendritic cells (iDCs) resulted in a significantly higher expression of DCs maturation markers (CD80, CD86 and HLA-DR) and induction of various cytokine and chemokine productions than in UVI-DENV-treated iDCs, suggesting a potential use of BCG- CS-NPs as adjuvant and delivery system for dengue vaccines.

A novel dengue virus serotype-2 nanovaccine induces robust humoral and cell-mediated immunity in mice.

Dengue virus (DENV), a member of the Flaviviridae family, can be transmitted to humans through the bite of infected Aedes mosquitoes. The incidence of dengue has increased worldwide over the past few decades. Inadequate vector control, changing global ecology, increased urbanization, and faster global travel are factors enhancing the rapid spread of the virus and its vector. In the absence of specific antiviral treatments, the search for a safe and effective vaccine grows more imperative. Many strategies have been utilized to develop dengue vaccines. Here, we demonstrate the immunogenic properties of a novel dengue nanovaccine (DNV), composed of ultraviolet radiation (UV)-inactivated DENV-2, which has been loaded into the nanoparticles containing chitosan/Mycobacterium bovis Bacillus Calmette-Guerin cell wall components (CS/BCG-NPs). We investigated the immunogenicity of DNV in a Swiss albino mouse model. Inoculation with various concentrations of vaccine (0.3, 1, 3 and 10µg/dose) with three doses, 15-day apart, induced strong anti-dengue IgM and IgG antibodies in the mouse serum along with neutralizing antibody against DENV-2 reference strain (16681), a clinical-isolate strain (00745/10) and the mouse-adapted New Guinea-C (NGC) strain. Cytokine and chemokine secretion in the serum of DNV-immunized mice showed elevated levels of IFN-γ, IL-2, IL-5, IL-12p40, IL-12p70, IL-17, eotaxin and RANTES, all of which have varying immune functions. Furthermore, we observed a DNV dose-dependent increase in the frequencies of IFN-γ-producing CD4(+) and CD8(+) T cells after in vitro stimulation of nucleated cells. Based on these findings, DNV has the potential to become a candidate dengue vaccine.

Genetic characterisation of a whiB7 mutant of a Mycobacterium tuberculosis clinical strain.

Mycobacterium tuberculosis is naturally resistant to clarithromycin (CLR). The genes Rv3197A (whiB7) and Rv1988 (ermMT) have been shown to be involved in the resistant phenotype. In this study, a CLR-susceptible M. tuberculosis clinical strain was identified, designated as DS3214, and the nucleotide sequences and expression profiles of whiB7 and ermMT were investigated. The results revealed that strain DS3214 contained a one nucleotide deletion in whiB7, leading to a truncated peptide. Expression of whiB7 was low, whereas comparable expression of ermMT was determined compared with the reference strain M. tuberculosis H37Rv. Overexpression of the mutant whiB7 in M. tuberculosis H37Ra did not increase the minimum inhibitory concentration (MIC) to CLR or kanamycin, indicating the defect of the mutant WhiB7. The CLR-susceptible M. tuberculosis clinical strain, whose whiB7 is naturally mutated, was first described in this study and whiB7 has been shown to play a role in the CLR-susceptible phenotype.

Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis.

BACKGROUND: Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is 'RHA' unlike 'RHG' found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. RESULTS: Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. CONCLUSION: Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic annotation systems towards a better understanding of this important group of enzymes.