Facile metabolic reprogramming distinguishes mycobacterial adaptation to hypoxia and starvation: ketosis drives starvation-induced persistence in M. bovis BCG.

Mycobacteria adapt to infection stresses by entering a reversible non-replicating persistence (NRP) with slow or no cell growth and broad antimicrobial tolerance. Hypoxia and nutrient deprivation are two well-studied stresses commonly used to model the NRP, yet little is known about the molecular differences in mycobacterial adaptation to these distinct stresses that lead to a comparable NRP phenotype. Here we performed a multisystem interrogation of the Mycobacterium bovis BCG (BCG) starvation response, which revealed a coordinated metabolic shift away from the glycolysis of nutrient-replete growth to depletion of lipid stores, lipolysis, and fatty acid ß-oxidation in NRP. This contrasts with BCG's NRP hypoxia response involving a shift to cholesterol metabolism and triglyceride storage. Our analysis reveals cryptic metabolic vulnerabilities of the starvation-induced NRP state, such as their newfound hypersensitivity to H2O2. These observations pave the way for developing precision therapeutics against these otherwise drug refractory pathogens.

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis.

BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection. RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice.

Current coronavirus disease 2019 vaccines face limitations including waning immunity, immune escape by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, limited cellular response, and poor mucosal immunity. We engineered a Clec9A-receptor binding domain (RBD) antibody construct that delivers the SARS-CoV-2 RBD to conventional type 1 dendritic cells. Compared with non-targeting approaches, single dose immunization in mice with Clec9A-RBD induced far higher RBD-specific antibody titers that were sustained for up to 21 months after vaccination. Uniquely, increasing neutralizing and antibody-dependent cytotoxicity activities across the sarbecovirus family was observed, suggesting antibody affinity maturation over time. Consistently and remarkably, RBD-specific follicular T helper cells and germinal center B cells persisted up to 12 months after immunization. Furthermore, Clec9A-RBD immunization induced a durable mono- and poly-functional T-helper 1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants of concern, including Omicron subvariants, and with a robust CD8+ T cell signature. Uniquely, Clec9A-RBD single-shot systemic immunization effectively primed RBD-specific cellular and humoral immunity in lung and resulted in significant protection against homologous SARS-CoV-2 challenge as evidenced by limited body weight loss and approximately 2 log10 decrease in lung viral loads compared with non-immunized controls. Therefore, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal protective immunity against rapidly evolving SARS-CoV2 variants.

Expanding the anti-flaviviral arsenal: Discovery of a baicalein-derived Compound with potent activity against DENV and ZIKV.

With approximately 3.8 billion people at risk of infection in tropical and sub-tropical regions, Dengue ranks among the top ten threats worldwide. Despite the potential for severe disease manifestation and the economic burden it places on endemic countries, there is a lack of approved antiviral agents to effectively treat the infection. Flavonoids, including baicalein, have garnered attention for their antimicrobial properties. In this study, we took a rational and iterative approach to develop a series of baicalein derivatives with improved antiviral activity against Dengue virus (DENV). Compound 11064 emerged as a promising lead candidate, exhibiting antiviral activity against the four DENV serotypes and representative strains of Zika virus (ZIKV) in vitro, with attractive selectivity indices. Mechanistic studies revealed that Compound 11064 did not prevent DENV attachment at the cell surface, nor viral RNA synthesis and viral protein translation. Instead, the drug was found to impair the post-receptor binding entry steps (endocytosis and/or uncoating), as well as the late stage of DENV infection cycle, including virus assembly/maturation and/or exocytosis. The inability to raise DENV resistant mutants, combined with significant antiviral activity against an unrelated RNA virus (Enterovirus-A71) suggested that Compound 11064 targets the host rather than a viral protein, further supporting its broad-spectrum antiviral potential. Overall, Compound 11064 represents a promising antiviral candidate for the treatment of Dengue and Zika.

Functionalized sulfonyl anthranilic acid derivatives inhibit replication of all the four dengue serotypes.

Dengue virus (DENV), a mosquito-borne flavivirus, continues to be a major public health threat in many countries and no approved antiviral therapeutics are available yet. In this work, we designed and synthesized a series of sulfonyl anthranilic acid (SAA) derivatives using a ligand-based scaffold morphing approach of the 2,1-benzothiazine 2,2-dioxide core, previously used by us to develop DENV polymerase inhibitors resulting devoid of any cell-based antiviral activity. Several derivatives based on the new SAA chemotype exhibited potent inhibition against DENV infection in the cell-based assay but did not inhibit DENV NS5 polymerase activity in the in vitro de novo initiation and elongation assays. Notably, best compounds 26 and 39 showed EC50 values in the range of 0.54-1.36 µM against cells infected with the four dengue serotypes (DENV-1-4). Time-of-drug-addition assay revealed that analogue 26 is a post-entry replication inhibitor that appears to be specific for cells of primate origin, implicating a host target with a high barrier to resistance. In conclusion, SAA derivatives offer a valuable starting point for developing effective Dengue antiviral therapeutics.

A Single Amino Acid Substitution in Structural Protein VP2 Abrogates the Neurotropism of Enterovirus A-71 in Mice.

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo, VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target.

A single-shot vaccine approach for the universal influenza A vaccine candidate M2e.

SignificanceAlthough the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24-amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.

Mitochondria-mediated oxidative stress during viral infection.

Through oxidative phosphorylation, mitochondria play a central role in energy production and are an important production source of reactive oxygen species (ROS). Not surprisingly, viruses have evolved to exploit this organelle in order to support their infection cycle. Beyond its role in the cellular antiviral response, induction of oxidative stress has emerged as a common strategy employed by many viruses to promote their replication. Here, we review the key molecular mechanisms employed by viruses to interact with mitochondria and induce oxidative stress. Furthermore, we discuss how viruses benefit from increased ROS levels, how they control ROS production to maintain a favorable redox environment, and how they cope with ROS-mediated cell death.

In vitro and in vivo efficacy of Metformin against dengue.

Dengue is a prevalent mosquito-borne viral infection in the tropical and sub-tropical regions. Its potential to progress into severe, life-threatening disease, has pressed the research community to develop safe, effective and affordable antivirals. Metformin (MET), a first-line antidiabetic drug and indirect AMP-activated protein kinase (AMPK) activator, has recently emerged as a potential anti-DENV therapeutic candidate, based on some experimental evidence supporting anti-DENV activity in vitro and widely reported anti-inflammatory properties. Here, we examined MET in vitro activity against the four DENV serotypes and in two different mammalian cell lines. MET displayed a poor anti-DENV activity in BHK-21 cells with IC50 in the mM range, which was associated with increased p-AMPKα levels, thereby supporting that MET antiviral activity is mediated through AMPK activation. In contrast, MET exerted a pro-DENV activity in Vero cells that did not correlate with increased AMPK activation, suggesting AMPK-independent effects. Treatment with compound 991, a direct AMPK activator, led to reduced viral titers against all four serotypes and across both mammalian cell lines. In vivo, oral administration of MET did not reduce viremia titers in an asymptomatic mouse model, neither did it improve disease severity and progression in a mouse model of severe dengue. Instead, high dose regimen worsened disease outcome as evidenced by increased mortality, higher viremia and hyper-inflammation. Therefore, while AMPK may represent a potential host target, MET does not seem to hold great promise as a pan-serotype anti-dengue drug.

Maternal Immunity and Vaccination Influence Disease Severity in Progeny in a Novel Mast Cell-Deficient Mouse Model of Severe Dengue.

Sub-neutralizing concentrations of antibodies in dengue infected patients is a major risk factor for the development of dengue hemorrhagic fever and dengue shock syndrome. Here, we describe a mouse model with a deficiency in mast cells (MCs) in addition to a deficiency in Type-I and II IFN receptors for studying dengue virus (DENV) infection. We used this model to understand the influence of MCs in a maternal antibody-dependent model of severe dengue, where offspring born to DENV-immune mothers are challenged with a heterologous DENV serotype. Mice lacking both MCs and IFN receptors were found susceptible to primary DENV infection and showed morbidity and mortality. When these mice were immunized, pups born to DENV-immune mothers were found to be protected for a longer duration from a heterologous DENV challenge. In the absence of MCs and type-I interferon signaling, IFN-γ was found to protect pups born to naïve mothers but had the opposite effect on pups born to DENV-immune mothers. Our results highlight the complex interactions between MCs and IFN-signaling in influencing the role of maternal antibodies in DENV-induced disease severity.

Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system.

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71.

Viral determinants that drive Enterovirus-A71 fitness and virulence.

Hand, Foot and Mouth Disease (HFMD) is usually a self-limiting, mild childhood disease that is caused mainly by Coxsackie virus A16 (CVA16) and Enterovirus A71 (EV-A71), both members of the Picornaviridae family. However, recurring HFMD outbreaks and epidemics due to EV-A71 infection in the Western Pacific region, and the propensity of EV-A71 strains to cause severe neurological complications have made this neurotropic virus a serious public health concern in afflicted countries. High mutation rate leading to viral quasispecies combined with frequent intra- and inter-typic recombination events amongst co-circulating EV-A71 strains have contributed to the great diversity and fast evolution of EV-A71 genomes, making impossible any accurate prediction of the next epidemic strain. Comparative genome sequence analyses and mutagenesis approaches have led to the identification of a number of viral determinants involved in EV-A71 fitness and virulence. These viral determinants include amino acid residues located in the structural proteins of the virus, affecting attachment to the host cell surface, receptor binding, and uncoating events. Critical residues in non-structural proteins have also been identified, including 2C, 3A, 3C proteases and the RNA-dependent RNA polymerase. Finally, mutations altering key secondary structures in the 5' untranslated region were also found to influence EV-A71 fitness and virulence. While our current understanding of EV-A71 pathogenesis remains fragmented, these studies may help in the rational design of effective treatments and broadly protective vaccine candidates.

HflX is a GTPase that controls hypoxia-induced replication arrest in slow-growing mycobacteria.

GTPase high frequency of lysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Escherichia coli. Here we report that HflX produced by slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, M. bovis BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between M. bovis BCG and M. tuberculosis, our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.

Changes in complement alternative pathway components, factor B and factor H during dengue virus infection in the AG129 mouse.

The complement alternative pathway (AP) is tightly regulated and changes in two important AP components, factor B (FB) and factor H (FH) are linked to severe dengue in humans. Here, a mouse model of dengue was investigated to define the changes in FB and FH and assess the utility of this model to study the role of the AP in severe dengue. Throughout the period of viremia in the AG129 IFN signalling-deficient mouse, an increase in FB and a decrease in FH was observed following dengue virus (DENV) infection, with the former only seen in a model of more severe disease associated with antibody-dependent enhancement (ADE). Terminal disease was associated with a decrease in FB and FH, with greater changes during ADE, and accompanied by increased C3 degradation consistent with complement activation. In silico analysis of NFκΒ, signal transducer and activator of transcription (STAT) and IFN-driven FB and FH promoter elements to reflect the likely impact of the lack of IFN-responses in AG129 mice, demonstrated that these elements differed markedly between human and mouse, notably with mouse FH lacking NFκΒ and key IFN-stimulated response elements (ISRE), and FB with many more NFκΒ and STAT-responsive elements than human FB. Thus, the AG129 mouse offers utility in demonstrating changes in FB and FH that, similar to humans, are associated with severe disease, but lack predicted important human-specific and IFN-dependent responses of FB and FH to DENV-infection that are likely to regulate the subtleties of the overall AP response during dengue disease in humans.

A single-dose live attenuated chimeric vaccine candidate against Zika virus.

The mosquito-borne Zika virus is an emerging pathogen from the Flavivirus genus for which there are no approved antivirals or vaccines. Using the clinically validated PDK-53 dengue virus vaccine strain as a backbone, we created a chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus. VacDZ demonstrates key markers of attenuation: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in interferon deficient adult AG129 mice. VacDZ may be administered as a traditional live virus vaccine, or as a DNA-launched vaccine that produces live VacDZ in vivo after delivery. Both vaccine formulations induce a protective immune response against Zika virus in AG129 mice, which includes neutralising antibodies and a strong Th1 response. This study demonstrates that VacDZ is a safe and effective vaccine candidate against Zika virus.

Sustainable, three-component, one-pot procedure to obtain active anti-flavivirus agents.

The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis, and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are available. Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral NS5 polymerase. However, synthesis of PBTZ analogues entails a high number of purification steps, the use of hazardous reagents and environmentally unsustainable generation of waste. Considering the promising antiviral activity of PBTZ analogues which require further exploration, in this work, we report the development of a new and sustainable three-component reaction (3CR) that can be combined with a basic hydrolysis in a one-pot procedure to obtain the PBTZ scaffold, thus reducing the number of synthetic steps, improving yields and saving time. 3CR was significantly explored in order to demonstrate its wide scope by using different starting materials. In addition, taking advantage of these procedures, we next designed and synthesized a new set of PBTZ analogues that were tested as anti-DENV-2 and anti-ZIKV agents. Compound 22 inhibited DENV-2 NS5 polymerase with an IC50 of 10.4 µM and represented the best anti-flavivirus compound of the new series by inhibiting DENV-2- and ZIKV-infected cells with EC50 values of 1.2 and 5.0 µM, respectively, that translates into attractive selectivity indexes (SI - 83 and 20, respectively). These results strongly reaffirm PBTZ derivatives as promising anti-flavivirus agents that now can be synthesized through a convenient and sustainable 3CR in order to obtain more potent compounds for further pre-clinical development studies.

An update on dengue vaccine development, challenges, and future perspectives.

INTRODUCTION: From both a public health and economic perspective, vaccination is arguably the most effective approach to combat endemic and pandemic infectious diseases. Dengue affects more than 100 countries in the tropical and subtropical world, with 100-400 million infections every year. In the wake of the recent setback faced by Dengvaxia, the only FDA-approved dengue vaccine, safer and more effective dengue vaccines candidates are moving along the clinical pipeline. AREA COVERED: This review provides an update of the latest outcomes of dengue vaccine clinical trials. In the light of recent progress made in our understanding of dengue pathogenesis and immune correlates of protection, novel vaccine strategies have emerged with promising second-generation dengue vaccine candidates. Finally, the authors discuss the dengue-specific challenges that remain to be addressed and overcome. EXPERT OPINION: The authors propose to explore various adjuvants and delivery systems that may help improve the design of safe, effective, and affordable vaccines against dengue. They also challenge the concept of a 'universal' dengue vaccine as increasing evidence support that DENV strains have evolved different virulence mechanisms.

Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis.

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F1 F0 ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa3 terminal oxidase. A functional redundancy between the cytochrome bcc:aa3 and the cytochrome bd oxidase protects M. tuberculosis from Q203-induced death, highlighting the attractiveness of the bd-type terminal oxidase for drug development. Here, we employed a facile whole-cell screen approach to identify the cytochrome bd inhibitor ND-011992. Although ND-011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic-tolerant, non-replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment.

Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.