CD137 expression and signal function drive pleiotropic γδ T-cell effector functions that inhibit intracellular M. tuberculosis growth.

Co-activation signal that induces/sustains pleiotropic effector functions of antigen-specific γδ T cells remains unknown. Here, Mycobacteria tuberculosis (Mtb) tuberculin administration during tuberculosis (TB) skin test resulted in rapid expression of co-activation signal molecules CD137 and CD107a by fast-acting Vγ2Vδ2 T cells in TB-resistant subjects (Resisters), but not patients with active TB. And, anti-CD137 agonistic antibody treatment experiments showed that CD137 signaling enabled Vγ2Vδ2 T cells to produce more effector cytokines and inhibit intracellular Mtb growth in macrophages (Mɸ). Consistently, Mtb antigen (Ag) HMBPP stimulation induced sustainable high-level CD137 expression in fresh and activated Vγ2Vδ2 T cells from uninfected subjects, but not TB patients. CD137+Vγ2Vδ2 T-cell subtype predominantly displayed central memory phenotype and mounted better proliferative responses than CD137-Vγ2Vδ2 T-cells. In response to HMBPP, CD137+Vγ2Vδ2 T-cell subtype rapidly differentiated into greater numbers of pleiotropic effector cells producing anti-Mtb cytokines compared to CD137-Vγ2Vδ2 T subtype, with the non-canonical NF-κB pathway involved. CD137 expression in Vγ2Vδ2 T cells appeared to signal anti-Mtb effector functions leading to intracellular Mtb growth inhibition in Mɸ, and active TB disrupted such CD137-driven anti-Mtb effector functions. CD137+Vγ2Vδ2 T-cells subtype exhibited an epigenetic-driven high-level expression of GM-CSF and de novo production of GM-CSF critical for Vγ2Vδ2 T-cell controlling of Mtb growth in Mϕ. Concurrently, exosomes produced by CD137+Vγ2Vδ2 T cells potently inhibited intracellular mycobacterial growth. Furthermore, adoptive transfer of human CD137+Vγ2Vδ2 T cells to Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, our data suggest that CD137 expression/signaling drives pleiotropic γδ T-cell effector functions that inhibit intracellular Mtb growth.

Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, is still one of the top killers worldwide among infectious diseases. The escape of Mtb from immunological clearance and the low targeting effects of anti-TB drugs remain the substantial challenges for TB control. Iron is particularly required for Mtb growth but also toxic for Mtb in high dosages, which makes iron an ideal toxic decoy for the 'iron-tropic' Mtb. Here, a macrophage-targeted iron oxide nanoparticles (IONPs)-derived IONPs-PAA-PEG-MAN nanodecoy is designed to augment innate immunological and drug killings against intracellular Mtb. IONPs-PAA-PEG-MAN nanodecoy exhibits preferential uptake in macrophages to significantly increase drug uptake with sustained high drug contents in host cells. Moreover, it can serve as a specific nanodecoy for the 'iron-tropic' Mtb to realize the localization of Mtb contained phagosomes surrounding the drug encapsulated nanodecoys and co-localization of Mtb with the drug encapsulated nanodecoys in lysosomes, where the incorporated rifampicin (Rif) can be readily released under acidic lysosomal condition for enhanced Mtb killing. This drug encapsulated nanodecoy can also polarize Mtb infected macrophages into anti-mycobacterial M1 phenotype and enhance M1 macrophage associated pro-inflammatory cytokine (TNF-α) production to trigger innate immunological responses against Mtb. Collectively, Rif@IONPs-PAA-PEG-MAN nanodecoy can synergistically enhance the killing efficiency of intracellular Mtb in in vitro macrophages and ex vivo monocyte-derived macrophages, and also significantly reduce the mycobacterial burdens in the lung of infected mice with alleviated pathology. These results indicate that Rif@IONPs-PAA-PEG-MAN nanodecoy may have a potential for the development of more effective therapeutic strategy against TB by manipulating augmented innate immunity and drug killings.

Adjunctive Zoledronate + IL-2 administrations enhance anti-tuberculosis Vγ2Vδ2 T-effector populations, and improve treatment outcome of multidrug-resistant tuberculosis1.

Multidrug-resistant tuberculosis (MDR-TB) is a refractory disease with high mortality rate due to no or few choices of antibiotics. Adjunctive immunotherapy may help improve treatment outcome of MDR-TB. Our decade-long studies demonstrated that phosphoantigen-specific Vγ2Vδ2 T cells play protective roles in immunity against TB. Here, we hypothesized that enhancing protective Vγ2Vδ2 T-effector cells could improve treatment outcome of MDR-TB. To address this, we employed clinically approved drugs Zoledronate (ZOL) and IL-2 to induce anti-TB Vγ2Vδ2 T-effector cells as adjunctive immunotherapy against MDR-TB infection of macaques. We found that adjunctive ZOL/IL-2 administrations during TB drugs treatment of MDR-TB-infected macaques significantly expanded Vγ2Vδ2 T cells and enhanced/sustained Vγ2Vδ2 T-effector subpopulation producing anti-TB cytokines until week 21. ZOL/IL-2 administrations, while expanding Vγ2Vδ2 T cells, significantly increased/sustained numbers of circulating CD4+ Th1 and CD8+ Th1-like effector populations, with some γδ T- or αß T-effector populations trafficking to airway at week 3 until week 19 or 21 after MDR-TB infection. Adjunctive ZOL/IL-2 administrations after MDR-TB infection led to lower bacterial burdens in lungs than TB drugs alone, IL-2 alone or saline controls, and resulted in milder MDR-TB pathology/lesions. Thus, adjunctive Zoledronate + IL-2 administrations can enhance anti-TB Vγ2Vδ2 T- and αß T-effector populations, and improve treatment outcome of MDR-TB.

Nanocages engineered from Bacillus Calmette-Guerin facilitate protective Vγ2Vδ2 T cell immunity against Mycobacterium tuberculosis infection.

Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a top killer among infectious diseases. While Bacillus Calmette-Guerin (BCG) is the sole TB vaccine, the clumped-clustered features of BCG in intradermal immunization appear to limit both the BCG protection efficacy and the BCG vaccination safety. We hypothesize that engineering of clumped-clustered BCG into nanoscale particles would improve safety and also facilitate the antigen-presenting-cell (APC)'s uptake and the following processing/presentation for better anti-TB protective immunity. Here, we engineered BCG protoplasts into nanoscale membraned BCG particles, termed as "BCG-Nanocage" to enhance the anti-TB vaccination efficiency and safety. BCG-Nanocage could readily be ingested/taken by APC macrophages selectively; BCG-Nanocage-ingested macrophages exhibited better viability and developed similar antimicrobial responses with BCG-infected macrophages. BCG-Nanocage, like live BCG bacilli, exhibited the robust capability to activate and expand innate-like T effector cell populations of Vγ2+ T, CD4+ T and CD8+ T cells of rhesus macaques in the ex vivo PBMC culture. BCG-Nanocage immunization of rhesus macaques elicited similar or stronger memory-like immune responses of Vγ2Vδ2 T cells, as well as Vγ2Vδ2 T and CD4+/CD8+ T effectors compared to live BCG vaccination. BCG-Nanocage- immunized macaques developed rapidly-sustained pulmonary responses of Vγ2Vδ2 T cells upon Mtb challenge. Furthermore, BCG- and BCG-Nanocage- immunized macaques, but not saline controls, exhibited undetectable Mtb infection loads or TB lesions in the Mtb-challenged lung lobe and hilar lymph node at endpoint after challenge. Thus, the current study well justifies a large pre-clinical investigation to assess BCG-Nanocage for safe and efficacious anti-TB vaccination, which is expected to further develop novel vaccines or adjuvants.

Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA-874-3p.

OBJECTIVES: Genetic and epigenetic mechanisms regulate antimicrobial immunity against Mycobacterium tuberculosis (Mtb) infection. METHODS: The present study assessed circular RNA TRAPPC6B (circTRAPPC6B) for antimicrobial immune functions and defined mechanisms wherein circTRAPPC6B regulates Mtb growth, autophagy and microRNA in macrophages. RESULTS: The Mtb infection of monocytes/macrophages resulted in a significantly decreased level of circTRAPPC6B that inhibited intracellular Mtb growth in macrophages. Conversely, circTRAPPC6B expression enhanced autophagy or autophagy-associated protein LC3-II production in Mtb-infected macrophages. circTRAPPC6B-enhanced autophagy aggregation or sequestration was also observed in fluorescence in situ hybridisation (FISH) analysis and confocal imaging. Mechanistically, circTRAPPC6B targets an inhibiting element miR-874-3p, as shown by bioinformatics, dual-luciferase reporter gene analysis and pull-down assay, respectively. Notably, miR-874-3p prohibited autophagy via suppressing autophagy protein ATG16L1 by binding to its 3'-untranslated region (UTR) in Mtb-infected macrophages and thus promoting intracellular Mtb growth. Concurrently, circTRAPPC6B enhanced autophagy in Mtb-infected macrophages by blocking the ability of miR-874-3p to inhibit ATG16L1. Thus, circTRAPPC6B antagonises the ability of miR-874-3p to suppress ATG16L1 expression and activate and enhance autophagy sequestration to restrict Mtb growth in macrophages. CONCLUSION: The current findings suggested that both circTRAPPC6B and miR-874-3p mechanisms can be explored as potential therapeutics against Mtb infection.

A CD4+CD161+ T-Cell Subset Present in Unexposed Humans, Not Tb Patients, Are Fast Acting Cells That Inhibit the Growth of Intracellular Mycobacteria Involving CD161 Pathway, Perforin, and IFN-γ/Autophagy.

It remains undefined whether a subset of CD4+ T cells can function as fast-acting cells to control Mycobacterium tuberculosis (Mtb) infection. Here we show that the primary CD4+CD161+ T-cell subset, not CD4+CD161-, in unexposed healthy humans fast acted as unconventional T cells capable of inhibiting intracellular Mtb and BCG growth upon exposure to infected autologous and allogeneic macrophages or lung epithelial A549 cells. Such inhibition coincided with the ability of primary CD4+CD161+ T cells to rapidly express/secrete anti-TB cytokines including IFN-γ, TNF-α, IL-17, and perforin upon exposure to Mtb. Mechanistically, blockades of CD161 pathway, perforin or IFN-γ by blocking mAbs abrogated the ability of CD4+CD161+ T cells to inhibit intracellular mycobacterial growth. Pre-treatment of infected macrophages with inhibitors of autophagy also blocked the CD4+CD161+ T cell-mediated growth inhibition of mycobacteria. Furthermore, adoptive transfer of human CD4+CD161+ T cells conferred protective immunity against mycobacterial infection in SCID mice. Surprisingly, CD4+CD161+ T cells in TB patients exhibited a loss or reduction of their capabilities to produce perforin/IFN-γ and to inhibit intracellular growth of mycobacteria in infected macrophages. These immune dysfunctions were consistent with PD1/Tim3 up-regulation on CD4+CD161+ T cells in active tuberculosis patients, and the blockade of PD1/Tim3 on this subset cells enhanced the inhibition of intracellular mycobacteria survival. Thus, these findings suggest that a fast-acting primary CD4+CD161+T-cell subset in unexposed humans employs the CD161 pathway, perforin, and IFN-γ/autophagy to inhibit the growth of intracellular mycobacteria, thereby distinguishing them from the slow adaptive responses of conventional CD4+ T cells. The presence of fast-acting CD4+CD161+ T-cell that inhibit mycobacterial growth in unexposed humans but not TB patients also implicates the role of these cells in protective immunity against initial Mtb infection.

Macrophage-Targeted Isoniazid-Selenium Nanoparticles Promote Antimicrobial Immunity and Synergize Bactericidal Destruction of Tuberculosis Bacilli.

Pathogenesis hallmarks for tuberculosis (TB) are the Mycobacterium tuberculosis (Mtb) escape from phagolysosomal destruction and limited drug delivery into infected cells. Several nanomaterials can be entrapped in lysosomes, but the development of functional nanomaterials to promote phagolysosomal Mtb clearance remains a big challenge. Here, we report on the bactericidal effects of selenium nanoparticles (Se NPs) against Mtb and further introduce a novel nanomaterial-assisted anti-TB strategy manipulating Ison@Man-Se NPs for synergistic drug-induced and phagolysosomal destruction of Mtb. Ison@Man-Se NPs preferentially entered macrophages and accumulated in lysosomes releasing Isoniazid. Surprisingly, Ison@Man-Se/Man-Se NPs further promoted the fusion of Mtb into lysosomes for synergistic lysosomal and Isoniazid destruction of Mtb. Concurrently, Ison@Man-Se/Man-Se NPs also induced autophagy sequestration of Mtb, evolving into lysosome-associated autophagosomal Mtb degradation linked to ROS-mitochondrial and PI3K/Akt/mTOR signaling pathways. This novel nanomaterial-assisted anti-TB strategy manipulating antimicrobial immunity and Mtb clearance may potentially serve in more effective therapeutics against TB and drug-resistant TB.

Mannosylated graphene oxide as macrophage-targeted delivery system for enhanced intracellular M.tuberculosis killing efficiency.

Tuberculosis (TB), caused by M.tuberculosis (Mtb), has become a top killer among infectious diseases. Enhancing the ability of anti-TB drugs to kill intracellular Mtb in host cells remains a big challenge. Here, an innovative nano-system was developed to increase drug delivery and Mtb-killing efficacy in Mtb-infected macrophages. We employed mannose surface decoration to develop mannosylated and PEGylated graphene oxide (GO-PEG-MAN). Such nano-platform exhibited increased uptake by macrophages via mannose receptor-mediated endocytosis in vitro. Interestingly, drug-loaded GO-PEG-MAN was preferentially up-taken by mannose receptor-expressing mucosal CD14+ macrophages isolated from Mtb-infected rhesus macaques than drug-loaded GO-PEG. Consistently, the drug concentration was also significantly higher in macrophages than that in T and B cells expressing no or low mannose receptor, implicating a useful macrophage/mannose receptor-targeted drug-delivery system relevant to the in vivo settings. Concurrently, rifampicin-loaded GO-PEG-MAN (Rif@GO-PEG-MAN) significantly increased rifampicin uptake, inducing long-lasting higher concentration of rifampicin in macrophages. Such innovative Rif@GO-PEG-MAN could readily get into the lysosomes of the Mtb host cells, where rifampicin underwent an accelerated release in acidic lysosomic condition, leading to explosive rifampicin release after cell entry for more effective killing of intracellular Mtb. Most importantly, Rif@GO-PEG-MAN-enhanced intracellular rifampicin delivery and pharmacokinetics significantly increased the efficacy of rifampicin-driven killing of intracellular BCG and Mtb bacilli in infected macrophages both in vitro and ex vivo. Such innovative nanocarrier approach may potentially enhance anti-TB drug efficacy and reduce drug side effects.

Immunization of Vγ2Vδ2 T cells programs sustained effector memory responses that control tuberculosis in nonhuman primates.

Tuberculosis (TB) remains a leading killer among infectious diseases, and a better TB vaccine is urgently needed. The critical components and mechanisms of vaccine-induced protection against Mycobacterium tuberculosis (Mtb) remain incompletely defined. Our previous studies demonstrate that Vγ2Vδ2 T cells specific for (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen are unique in primates as multifunctional effectors of immune protection against TB infection. Here, we selectively immunized Vγ2Vδ2 T cells and assessed the effect on infection in a rhesus TB model. A single respiratory vaccination of macaques with an HMBPP-producing attenuated Listeria monocytogenes (Lm ΔactA prfA*) caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in circulating and pulmonary compartments. This did not occur in animals similarly immunized with an Lm ΔgcpE strain, which did not produce HMBPP. Lm ΔactA prfA* vaccination elicited increases in Th1-like Vγ2Vδ2 T cells in the airway, and induced containment of TB infection after pulmonary challenge. The selective immunization of Vγ2Vδ2 T cells reduced lung pathology and mycobacterial dissemination to extrapulmonary organs. Vaccine effects coincided with the fast-acting memory-like response of Th1-like Vγ2Vδ2 T cells and tissue-resident Vγ2Vδ2 effector T cells that produced both IFN-γ and perforin and inhibited intracellular Mtb growth. Furthermore, selective immunization of Vγ2Vδ2 T cells enabled CD4+ and CD8+ T cells to mount earlier pulmonary Th1 responses to TB challenge. Our findings show that selective immunization of Vγ2Vδ2 T cells can elicit fast-acting and durable memory-like responses that amplify responses of other T cell subsets, and provide an approach to creating more effective TB vaccines.

Multidrug-resistant tuberculosis (MDR-TB) strain infection in macaques results in high bacilli burdens in airways, driving broad innate/adaptive immune responses.

Tuberculosis (TB) has become the most deadly infectious diseases due to epidemics of HIV/AIDS and multidrug-resistant/extensively drug-resistant TB (MDR-/XDR-TB). Although person-to-person transmission contributes to MDR-TB, it remains unknown whether infection with MDR strains resembles infection with drug-sensitive (DS) TB strains, manipulating limited or broad immune responses. To address these questions, macaques were infected with MDR strain V791 and a drug-sensitive Erdman strain of TB. MDR bacilli burdens in the airway were significantly higher than those of the Erdman control after pulmonary exposure. This productive MDR strain infection upregulated the expression of caspase 3 in macrophages/monocytes and induced appreciable innate-like effector responses of CD3-negative lymphocytes and Ag-specific γδ T-cell subsets. Concurrently, MDR strain infection induced broad immune responses of T-cell subpopulations producing Th1, Th17, Th22, and CTL cytokines. Furthermore, MDR bacilli, like the Erdman strain, were capable of inducing typical TB disease characterized by weight loss, lymphocytopenia, and severe TB lesions. For the first time, our results suggest that MDR-TB infection acts like DS to induce high bacterial burdens in the airway (transmission advantage), innate/adaptive immune responses, and disease processes. Because nonhuman primates are biologically closer to humans than other species, our data may provide useful information for predicting the effects of primary MDR strain infection after person-to-person transmission. The findings also support the hypothesis that a vaccine or host-directed adjunctive modality that is effective for drug-sensitive TB is likely to also impact MDR-TB.

Assessment of recombinant plasmid expressing fusion antigen Ag85B-Rv3425 in management of acute tuberculosis infection in mice.

The emergence of drug-resistant tuberculosis (TB) and HIV-TB co-infection fuels an urgent need to develop novel therapeutic approaches, including therapeutic vaccines. Therapeutic vaccines have been proven to be a good strategy by inducing antigen specific immune responses against TB infection. In the present study, a recombinant plasmid based on lentiviral vector expressing fusion antigen Ag85B-Rv3425 (A3), and was constructed the immunogenicity and treatment effects in TB mice were assessed. The results showed that A3 delivered by the plasmid could be expressed appropriately in vivo and induced higher production of tumor necrosis factor-α and interleukin-2 compared with A3 recombinant protein in mice. Moreover, the recombinant plasmid expressing A3 confered resistance to acute TB infection in mice, characterized by a reduction in the bacterial load in the lungs and spleen, as well as attenuated TB lesions in lung tissues. These results implicated that the recombinant plasmid based on lentiviral vector expressing A3 is a potent and promising therapeutic agent to treat acute TB infection.

IL-12+IL-18 Cosignaling in Human Macrophages and Lung Epithelial Cells Activates Cathelicidin and Autophagy, Inhibiting Intracellular Mycobacterial Growth.

The ability of Mycobacterium tuberculosis to block host antimicrobial responses in infected cells provides a key mechanism for disease pathogenesis. The immune system has evolved to overcome this blockade to restrict the infection, but it is not clear whether two key innate cytokines (IL-12/IL-18) involved in host defense can enhance antimycobacterial mechanisms. In this study, we demonstrated that the combination of IL-12 and IL-18 triggered an antimicrobial response against mycobacteria in infected macrophages (THP-1 and human primary monocyte-derived macrophages) and pulmonary epithelial A549 cells. The inhibition of intracellular bacterial growth required p38-MAPK and STAT4 pathways, the vitamin D receptor, the vitamin D receptor-derived antimicrobial peptide cathelicidin, and autophagy, but not caspase-mediated apoptosis. Finally, the ability of IL-12+IL-18 to activate an innate antimicrobial response in human primary macrophages was dependent on the autonomous production of IFN-γ and the CAMP/autophagy pathway. Together, these data suggest that IL-12+IL-18 cosignaling can trigger the antimicrobial protein cathelicidin and autophagy, resulting in inhibition of intracellular mycobacteria in macrophages and lung epithelial cells.

Recombinant Bacille Calmette-Guérin coexpressing Ag85B-IFN-γ enhances the cell-mediated immunity in C57BL/6 mice.

The only available vaccine against pulmonary tuberculosis is Bacille Calmette-Guérin (BCG). As the efficacy reported of the vaccine is not up to the mark, there is an urgent need to develop improved anti-tuberculosis vaccines. Antigen 85B (Ag85B) is a very promising vaccine candidate molecule of Mycobacterium tuberculosis and interferon (IFN)-γ and has been considered the most attractive correlate of protective immunity. The aim of this study was to construct a novel recombinant BCG (rBCG) to secrete Ag85B and mouse IFN-γ under control of the Mycobacterial heat shock protein 60 (hsp60) promoter and the antigen signal sequence. Second aim of the present study is to evaluate the immune response in C57BL/6 elicted by the new rBCG. Expression of the fusion protein was readily detectable by western blotting and IFN-γ bioactivity was detected indirectly by enzyme-linked immunosorbent assay (ELISA). Compared with BCG, rBCG::Ag85B-IFN-γ was substantially more active in inducing the production of IFN-γ and tumor necrosis factor (TNF)-α from mouse splenocytes. ELISA analysis for IgG, IgG1 and IgG2c showed that rBCG::Ag85B-IFN-γ induced higher titer of Ag85B and facilitated Th1 type immune response. rBCG::Ag85B-IFN-γ also improved nitric oxide production levels and enhanced antigen-specific splenocyte proliferation. Moreover, rBCG::Ag85B-IFN-γ induced human monocytes such as THP-1 cells to enhance expression of CD80, CD86, CD40 and HLA-DR. Flow cytometry analysis confirmed that rBCG::Ag85B-IFN-γ significantly activated CD4+ T cells. Assessing combinations of IFN-γ, TNF-α and interleukin-2 at the single-cell level by multiparameter flow cytometry, we found that rBCG::Ag85B-IFN-γ improved the multifunctional T cells level in comparison to BCG. In conclusion, the present study indicates that rBCG::Ag85B-IFN-γ increases cell mediated immune response and is a potential candidate vaccine for immunotherapeutic protocols against pulmonary tuberculosis.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

Ag85B synergizes with ESAT-6 to induce efficient and long-term immunity of C57BL/6 mice primed with recombinant Bacille Calmette-Guerin.

The latest probable scenario in vaccination strategies is to prime one live attenuated vaccine candidate followed by boost dose of second vaccine candidate. In the present study, we primed the mice with a recombinant Bacille Calmette-Guerin (BCG) comprising Ag85B and ESAT-6 followed by boost doses of Ag85B, ESAT-6 and Ag85B-ESAT-6 fusion protein in the DDA adjuvant, separately. After boost doses of 8 and 12 weeks, the levels of antigen-stimulated T cells secreting interferon (IFN)-γ, the content of the IFN-γ, tumor necrosis factor-α and interleukin-4 in the splenocytes in vitro culture supernatant, the antigen-specific immunoglobulin (Ig)G titer from mouse serum, IgG subclass and the population of antigen-specific CD4+ and CD8+ T cells were detected. The present study showed that recombinant BCG along with boost doses of Ag85B or ESAT-6 individually did not induce efficient T-helper (Th) 1-type immune response. On the other hand, recombinant BCG with boost doses of Ag85B-ESAT-6 fusion protein enhanced longer lasting predominant Th1 immune response. This result suggested that Ag85B might synergize with ESAT-6 protein in order to produce better as well as effective immune response. Thus, the present study concluded recombinant BCG with boost doses of Ag85B-ESAT-6 fusion protein could be a good strategy to improve the immune protective efficacy.

Cell-Based High-Throughput Screening Assay Identifies 2',2'-Difluoro-2'-deoxycytidine Gemcitabine as a Potential Antipoliovirus Agent.

As we approach the global eradication of circulating wild-type polioviruses (PV), vaccination with oral poliovirus vaccine (OPV) has led to the emergence of circulating vaccine-derived poliovirus (cVDPV) and vaccine-associated paralytic poliomyelitis (VAPP). Complete cessation of all poliovirus infections may require stopping use of OPV and formulating improved vaccines and new antiviral drugs. Currently, no licensed drugs are available to treat chronically infected poliovirus excretors. Here, we created a modified PV expressing Gaussia Luciferase (Sb-Gluc) and developed a cell-based high-throughput screening (HTS) antiviral assay. Using the validated HTS assay, we screened the FDA-approved drug library of compounds and identified candidate agents capable of inhibiting PV replication. We then characterized antipoliovirus activity for the best hit, gemcitabine, a nucleoside analogue used in tumor chemotherapy. We found that gemcitabine inhibited PV Mahoney replication with an IC50 of 0.3 µM. It completely protected HeLa cells from PV-induced cytopathic effects at 25 µM, without detectable toxicity for cell viability. Furthermore, a gemcitabine metabolite directly inhibited the ability of PV RNA polymerase to synthesize or elongate PV RNA. Because PV RNA polymerase is somehow conserved among species in the Picornaviridae family, gemcitabine may be further developed as an attractive broad-spectrum antiviral for PV and others.

Selective Destruction of Interleukin 23-Induced Expansion of a Major Antigen-Specific γδ T-Cell Subset in Patients With Tuberculosis.

A loss of antigen-specific T-cell responses due to defective cytokine signaling during infections has not been reported. We hypothesize that tuberculosis can destroy signaling effects of selective cytokine(s) and induce exhaustion of antigen-specific T cells. To test this hypothesis, mechanistic studies were performed to examine whether and how tuberculosis blocked interleukin 23 (IL-23) and interleukin 2 (IL-2) signaling effects on a major human γδ T-cell subpopulation, phosphoantigen HMBPP-specific Vγ2Vδ2 T cells. IL-23 and IL-2 significantly expanded HMBPP-stimulated Vγ2Vδ2 T cells from subjects with latent tuberculosis infection, and IL-2 synergized the effect of IL-23. IL-23-induced expansion of Vγ2Vδ2 T cells involved STAT3. Surprisingly, patients with tuberculosis exhibited a selective destruction of IL-23-induced expansion of these cells. The tuberculosis-driven destruction of IL-23 signaling coincided with decreases of expression and phosphorylation of STAT3. Interestingly, impairing of STAT3 was linked to marked increases in the microRNAs (miRNAs) hsa-miR-337-3p and hsa-miR-125b-5p in Vγ2Vδ2 T cells from patients with tuberculosis. Downregulation of hsa-miR-337-3p and hsa-miR-125b-5p by miRNA sponges improved IL-23-mediated expansion of Vγ2Vδ2 T cells and restored the ability of these cells to produce anti-tuberculosis cytokines. These results support our hypothesis that tuberculosis can selectively impair a cytokine effect while sparing another and can induce exhaustion of T cells in response to the respective cytokine.

Recombinant BCG prime and PPE protein boost provides potent protection against acute Mycobacterium tuberculosis infection in mice.

Since BCG, the only vaccine widely used against tuberculosis (TB) in the world, provides varied protective efficacy and may not be effective for inducing long-term cellular immunity, it is in an urgent need to develop more effective vaccines and more potent immune strategies against TB. Prime-boost is proven to be a good strategy by inducing long-term protection. In this study, we tested the protective effect against Mycobacterium tuberculosis (Mtb) challenge of prime-boost strategy by recombinant BCG (rBCG) expressing PPE protein Rv3425 fused with Ag85B and Rv3425. Results showed that the prime-boost strategy could significantly increase the protective efficiency against Mtb infection, characterized by reduction of bacterial load in lung and spleen, attenuation of tuberculosis lesions in lung tissues. Importantly, we found that Rv3425 boost, superior to Ag85B boost, provided better protection against Mtb infection. Further research proved that rBCG prime-Rv3425 boost could obviously increase the expansion of lymphocytes, significantly induce IL-2 production by lymphocytes upon PPD stimulation, and inhibit IL-6 production at an early stage. It implied that rBCG prime-Rv3425 boost opted to induce Th1 immune response and provided a long-term protection against TB. These results implicated that rBCG prime-Rv3425 boost is a potent and promising strategy to prevent acute Mtb infection.