MDA5 RNA-sensing pathway activation by Mycobacterium tuberculosis promotes innate immune subversion and pathogen survival.

Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis pathogenesis, we evaluated M. tuberculosis intracellular growth and innate immune responses in WT and Mda5-/- macrophages. Transfection of M. tuberculosis RNA strongly induced proinflammatory cytokine production in WT macrophages, which was abrogated in Mda5-/- macrophages. M. tuberculosis infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFN-γ-primed Mda5-/- macrophages effectively contained intracellular M. tuberculosis proliferation to a markedly greater degree than WT macrophages. Further comparisons of WT versus Mda5-/- macrophages revealed that during M. tuberculosis infection MDA5 contributed to IL-1ß production and inflammasome activation and that loss of MDA5 led to a substantial increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M. tuberculosis bacillary burden. These data reveal that loss of MDA5 is host protective during M. tuberculosis infection in vitro and in vivo, suggesting that M. tuberculosis exploits MDA5 to subvert immune containment.

Intravenous BCG vaccination reduces SARS-CoV-2 severity and promotes extensive reprogramming of lung immune cells.

Bacillus Calmette-Guérin (BCG) confers heterologous immune protection against viral infections and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here, we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model. BCG vaccination conferred a modest reduction on lung SCV2 viral load, bronchopneumonia scores, and weight loss, accompanied by a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. BCG uniquely recruited immunoglobulin-producing plasma cells to the lung suggesting accelerated local antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, with a transcriptional shift away from exhaustion markers and toward antigen presentation and repair. Similarly, BCG enhanced recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, that show reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Nucleolin mediates SARS-CoV-2 replication and viral-induced apoptosis of host cells.

Host-oriented antiviral therapeutics are promising treatment options to combat COVID-19 and its emerging variants. However, relatively little is known about the cellular proteins hijacked by SARS-CoV-2 for its replication. Here we show that SARS-CoV-2 induces expression and cytoplasmic translocation of the nucleolar protein, nucleolin (NCL). NCL interacts with SARS-CoV-2 viral proteins and co-localizes with N-protein in the nucleolus and in stress granules. Knockdown of NCL decreases the stress granule component G3BP1, viral replication and improved survival of infected host cells. NCL mediates viral-induced apoptosis and stress response via p53. SARS-CoV-2 increases NCL expression and nucleolar size and number in lungs of infected hamsters. Inhibition of NCL with the aptamer AS-1411 decreases viral replication and apoptosis of infected cells. These results suggest nucleolin as a suitable target for anti-COVID therapies.

Quantification of Infectious SARS-CoV-2 by the 50% Tissue Culture Infectious Dose Endpoint Dilution Assay.

A number of viral quantification methods are used to measure the concentration of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the traditional plaque-based assay allows for direct enumeration of replication competent lytic virions and remains the gold standard for the quantification of infectious virus, the 50% tissue culture infectious dose (TCID50) endpoint dilution assay allows for a more rapid, large-scale analysis of experimental samples. In this chapter, we describe a well-established TCID50 assay protocol to measure the SARS-CoV-2 infectious titer in viral stocks, in vitro cell or organoid models, and animal tissue. We also present alternative assays for scoring the cytopathic effect of SARS-CoV-2 in cell culture and comparable methods to calculate the 50% endpoint by serial dilution.

Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation.

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guérin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Sulforaphane exhibits in vitro and in vivo antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are no orally available medications for prophylaxis for those exposed to SARS-CoV-2 and limited therapeutic options for those who develop COVID-19. We evaluated the antiviral activity of sulforaphane (SFN), a naturally occurring, orally available, well-tolerated, nutritional supplement present in high concentrations in cruciferous vegetables with limited side effects. SFN inhibited in vitro replication of four strains of SARS-CoV-2 as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN is a promising treatment for prevention of coronavirus infection or treatment of early disease.

Allogeneic bone marrow transplantation with post-transplant cyclophosphamide for patients with HIV and haematological malignancies: a feasibility study.

BACKGROUND: Allogeneic blood or marrow transplantation (alloBMT) is a potentially life-saving treatment for individuals with HIV and haematological malignancies; challenges include identifying donors and maintaining antiretroviral therapy (ART). The objectives of our study were to investigate interventions to expand donor options and to prevent ART interruptions for patients with HIV in need of alloBMT. METHODS: This single-arm, interventional trial took place at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center (Baltimore, MD, USA). Individuals with HIV who were at least 18 years of age and referred for alloBMT for a standard clinical indication were eligible. The only exclusion criterion was a history of documented resistance to enfuvirtide. We used post-transplant cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis to expand donor options and an optimised ART strategy of avoiding pharmacoenhancers and adding subcutaneous enfuvirtide during post-transplant cyclophosphamide and during oral medication intolerance. Our primary outcome was the proportion of participants who maintained ART through day 60 after alloBMT. We measured the HIV latent reservoir using a quantitative viral outgrowth assay. This study is registered on ClinicalTrials.gov, NCT01836068. FINDINGS: Between June 1, 2013, and August 27, 2015, nine patients who were referred for transplant provided consent. Two patients had relapsed malignancy before donor searches were initiated. Seven patients had suitable donors identified (two matched sibling, two matched unrelated, two haploidentical, and one single-antigen mismatched unrelated) and proceeded to alloBMT. All patients maintained ART through day 60 and required ART changes (median 1, range 1-3) in the first 90 days. One patient stopped ART and developed HIV rebound with grade 4 meningoencephalitis at day 146. Among six patients who underwent alloBMT and had longitudinal measurements available, the HIV latent reservoir was not detected post-alloBMT in four patients with more than 95% donor chimerism, consistent with a 2·06-2·54 log10 reduction in the HIV latent reservoir. In the two patients with less than 95% donor chimerism, the HIV latent reservoir remained stable. INTERPRETATION: By using post-transplant cyclophosphamide as GVHD prophylaxis, we successfully expanded alloBMT donor options for patients with HIV. Continuing ART with a regimen that includes enfuvirtide post-alloBMT was safe, but life-threatening viral rebound can occur with ART interruption. FUNDING: amfAR (the Foundation for AIDS Research), Johns Hopkins University Center for AIDS Research, and National Cancer Institute.

Infectability of human BrainSphere neurons suggests neurotropism of SARS-CoV-2

Reports from Wuhan suggest that 36% of COVID-19 patients show neurological symptoms, and cases of viral encephalitis have been reported, suggesting that the virus is neurotropic under unknown circumstances. This is well established for other coronaviruses. In order to understand why some patients develop such symptoms and others do not, we address herein the infectability of the central nervous system (CNS). Reports that the ACE2 receptor ­ critical for virus entry into lung cells ­ is found in different neurons support this expectation. We employed a human induced pluripotent stem cell (iPSC)- derived BrainSphere model, which we used earlier for Zika, Dengue, HIV and John Cunningham virus infection studies. We detected the expression of the ACE2 receptor, but not TMPRSS2, in the model. Incubating the BrainSpheres for 6 hours with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 led to infection of a fraction of neural cells with replication of the virus evident at 72 hpi. Virus particles were found in the neuronal cell body extending into apparent neurite structures. PCR measurements corroborated the replication of the virus, suggesting at least a tenfold increase in virus copies per total RNA. Leveraging state-of-the-art 3D organotypic cell culture, which has been shown to allow both virus infection and modeling of (developmental) neurotoxicity but is at the same time simple enough to be transferred and used in a BSL-3 environment, we demonstrate, for the first time, the potential critically important neurotropism of SARS-CoV-2.

Relative and Quantitative Phosphoproteome Analysis of Macrophages in Response to Infection by Virulent and Avirulent Mycobacteria Reveals a Distinct Role of the Cytosolic RNA Sensor RIG-I in Mycobacterium tuberculosis Pathogenesis.

Comparative phosphoproteomics of Mycobacterium tuberculosis (Mtb)- and Mycobacterium bovis BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-ß, IL-1α, and IL-1ß levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013171.

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

Characterization of Elite Suppressors Cell-Associated HIV-1 mRNA at Baseline and with T Cell Activation

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Objective: Elite Controllers or Suppressors (ES) are patients who control HIV replication without antiretroviral therapy. In this study, we compared baseline and inducible HIV-1 mRNA levels in CD4+ T cells from ES and chronic progressors (CPs) receiving suppressive antiretroviral therapy. Methods: We quantified basal levels of cell associated HIV-1 mRNA in CD4+ T cells isolated from CPs and ES. Additionally, we measured the fold upregulation of intracellular HIV-mRNA after stimulation of CD4+ T cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and quantified the amount of HIV-mRNA levels released into culture supernatant. Results: ES have significantly less cell associated HIV-mRNA per 5x106 cells (p = 0.003); 8 of 10 CPs had quantifiable HIV-1 mRNA at baseline, whereas this was present in only 2 of 10 ES. Upon stimulation with PMA and ionomycin, 4 of 5 CPs and 7 of 9 ES showed increased cell associated HIV-mRNA. Interestingly, released HIV-1 mRNA could be detected in supernatants of CD4+ T cells stimulated with PMA/ionomycin from 5 of 8 ES. Conclusion: Our results demonstrate that while the baseline levels of cell associated HIV-1 mRNA are significantly lower in ES compared to CPs, stimulation of CD4+ T cells results in a comparable relative upregulation of viral transcription.

Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations.

Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs.

A primary CD4(+) T cell model of HIV-1 latency established after activation through the T cell receptor and subsequent return to quiescence.

A mechanistic understanding of HIV-1 latency depends on a model system that recapitulates the in vivo condition of latently infected, resting CD4(+) T lymphocytes. Latency seems to be established after activated CD4(+) T cells, the principal targets of HIV-1 infection, become productively infected and survive long enough to return to a resting memory state in which viral expression is inhibited by changes in the cellular environment. This protocol describes an ex vivo primary cell system that is generated under conditions that reflect the in vivo establishment of latency. Creation of these latency model cells takes 12 weeks and, once established, the cells can be maintained and used for several months. The resulting cell population contains both uninfected and latently infected cells. This primary cell model can be used to perform drug screens, to study cytolytic T lymphocyte (CTL) responses to HIV-1, to compare viral alleles or to expand the ex vivo life span of cells from HIV-1-infected individuals for extended study.

Protecting children from taking up smoking: parents' views on what would help.

ISSUE ADDRESSED: The present study investigated what factors the parents of children in low-income areas of Auckland, New Zealand, thought could help protect their children from smoking initiation. METHODS: Participants in a large quasi-experimental trial that tested a community-, school- and family-based smoking-initiation intervention were asked in a questionnaire 'What could we do to help you protect your children from smoke and taking up smoking?' Free-text responses were divided into distinct meaning units and categorised independently by two of the researchers. RESULTS: 1806 participants (70% of parents who returned the questionnaire) completed the question. The majority of respondents (80%) were either Pacific Island or Maori mothers and 25% were current smokers. Five main categories of suggested strategies for preventing smoking initiation were identified: building children's knowledge of the ill-effects of smoking; denormalising smoking; reducing access to tobacco; building children's resilience; and health promotion activities. The most common suggestion was to educate children about smoking. CONCLUSION: Building children's knowledge of smoking risks was the main strategy parents proposed. There was some support for banning smoking in most public areas and for tougher moves to stop tobacco sales to minors. Few parents suggested innovative or radical strategies, such as banning the sale of tobacco, fining children for smoking or use of competitions. So what? To ensure reductions in smoking initiation for lower socioeconomic and Maori and Pacific Island people, further research should engage Maori, Pacific Island and lower socioeconomic parents in a process that elicits innovative thinking about culturally acceptable strategies.

New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo.

HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART). This reservoir is the major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually.

Trends in cervical cancer in Fiji, 2000-2010.

SETTING: Cancer patients recorded in Fiji's National Patient Information System (PATIS) from 2000 to 2010. OBJECTIVE: To identify trends in cervical cancer using case numbers, incidence rates and case fatality in Fiji over the decade 2000-2010. DESIGN: Retrospective descriptive and analytical study. RESULTS: Between 2000 and 2010, 1234 patients were registered with cervical cancer, of whom 845 (68%) were indigenous Fijians and 357 (29%) were Indians; only 32 (3%) were of other ethnic groups. Mortality rates were much higher among Fijian women, and were far higher in women aged ≥45 years. CONCLUSION: The high incidence rates of cervical cancer in Fijian women between the ages of 35 and 45 years reflect ethnic differences in social norms. The higher case fatality and mortality rates in these groups indicate that more work is needed to improve access to and quality of screening and treatment services.

Primary school compliance with school canteen guidelines in Fiji and its association with student obesity.

SETTING: Childhood obesity is of growing public health concern in Fiji. The study setting was primary schools in Fiji's Western Division. OBJECTIVE: 1) To assess primary schools' compliance with national school canteen guidelines, 2) to understand reasons for non-compliance, and 3) to assess the relationship between compliance with the guidelines and students' body mass index (BMI). DESIGN: Cross-sectional analysis of data collected in 2010 by public health dieticians of the Ministry of Health on annual visits to primary schools. RESULTS: Among 230 schools, 33 (14%) had no canteen data. Of the 197 schools with data, only 31 (16%) were fully compliant with national school canteen guidelines, while the remaining 166 (84%) did not fully comply with the guidelines. This was irrespective of school location or whether the canteen was school or commercially operated. In a random sample (n = 44 schools), overweight and obesity were more common among children in non-compliant schools than in fully compliant schools (40% vs. 32%, P < 0.001). CONCLUSION: Most primary schools in Fiji's Western Division did not comply with school canteen guidelines, which is worrying given the increasing rates of overweight children. Given the association between non-compliance and student overweight/obesity, further action is needed to ensure that these guidelines are implemented.