Advancing Diagnosis and Treatment in People Living with HIV and Tuberculosis Meningitis.

PURPOSE OF REVIEW: Tuberculous meningitis (TBM) is the most severe form of tuberculosis. Inadequate diagnostic testing and treatment regimens adapted from pulmonary tuberculosis without consideration of the unique nature of TBM are among the potential drivers. This review focuses on the progress being made in relation to both diagnosis and treatment of TBM, emphasizing promising future directions. RECENT FINDINGS: The molecular assay GeneXpert MTB/Rif Ultra has improved sensitivity but has inadequate negative predictive value to "rule-out" TBM. Evaluations of tests focused on the host response and bacterial components are ongoing. Clinical trials are in progress to explore the roles of rifampin, fluoroquinolones, linezolid, and adjunctive aspirin. Though diagnosis has improved, novel modalities are being explored to improve the rapid diagnosis of TBM. Multiple ongoing clinical trials may change current therapies for TBM in the near future.

A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication.

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 µM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1-2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.

Anti-schistosomal immunity to core xylose/fucose in N-glycans.

Schistosomiasis is a globally prevalent, debilitating disease that is poorly controlled by chemotherapy and for which no vaccine exists. While partial resistance in people may develop over time with repeated infections and treatments, some animals, including the brown rat (Rattus norvegicus), are only semi-permissive and have natural protection. To understand the basis of this protection, we explored the nature of the immune response in the brown rat to infection by Schistosoma mansoni. Infection leads to production of IgG to parasite glycoproteins with complex-type N-glycans that contain a non-mammalian-type modification by core α2-Xylose and core α3-Fucose (core Xyl/Fuc). These epitopes are expressed on the surfaces of schistosomula and adult worms. Importantly, IgG to these epitopes can kill schistosomula by a complement-dependent process in vitro. Additionally, sera from both infected rhesus monkey and infected brown rat were capable of killing schistosomula in a manner inhibited by glycopeptides containing core Xyl/Fuc. These results demonstrate that protective antibodies to schistosome infections in brown rats and rhesus monkeys include IgG responses to the core Xyl/Fuc epitopes in surface-expressed N-glycans, and raise the potential of novel glyco-based vaccines that might be developed to combat this disease.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19.

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19.

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated sixteen of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

An 'Arms Race' between the Nonsense-mediated mRNA Decay Pathway and Viral Infections.

The Nonsense-mediated mRNA Decay (NMD) pathway is an RNA quality control pathway conserved among eukaryotic cells. While historically thought to predominantly recognize transcripts with premature termination codons, it is now known that the NMD pathway plays a variety of roles, from homeostatic events to control of viral pathogens. In this review we highlight the reciprocal interactions between the host NMD pathway and viral pathogens, which have shaped both the host antiviral defense and viral pathogenesis.

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

Central Nervous System Virus Infection in African Children with Cerebral Malaria.

We aimed to identify the contribution of central nervous system (CNS) viral coinfection to illness in African children with retinopathy-negative or retinopathy-positive cerebral malaria (CM). We collected cerebrospinal fluid (CSF) from 272 children with retinopathy-negative or retinopathy-positive CM and selected CSF from 111 of these children (38 retinopathy positive, 71 retinopathy negative, 2 retinopathy unknown) for analysis by metagenomic next-generation sequencing. We found CSF viral coinfections in 7/38 (18.4%) retinopathy-positive children and in 18/71 (25.4%) retinopathy-negative children. Excluding HIV-1, human herpesviruses (HHV) represented 61% of viruses identified. Excluding HIV-1, CNS viral coinfection was equally likely in children who were retinopathy positive and retinopathy negative (P = 0.1431). Neither mortality nor neurological morbidity was associated with the presence of virus (odds ratio [OR] = 0.276, 95% CI: 0.056-1.363). Retinopathy-negative children with a higher temperature, lower white blood cell count, or being dehydrated were more likely to have viral coinfection. Level of consciousness at admission was not associated with CNS viral coinfection in retinopathy-negative children. Viral CNS coinfection is unlikely to contribute to coma in children with CM. The herpesviruses other than herpes simplex virus may represent incidental bystanders in CM, reactivating during acute malaria infection.

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis.

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.

The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein.

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1). Endogenous UPF1 interacted with the ZIKV capsid protein in coimmunoprecipitation experiments, and capsid expression posttranscriptionally downregulated UPF1 protein levels, a process that we confirmed occurs during ZIKV infection. Cellular fractionation studies show that the ZIKV capsid protein specifically targets nuclear UPF1 for degradation via the proteasome. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures, consistent with a model in which NMD restricts ZIKV infection in the fetal brain. We propose that ZIKV, via the capsid protein, has evolved a strategy to lower UPF1 levels and dampen antiviral activities of NMD, which in turn contributes to neuropathology in vivoIMPORTANCE Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.

Fatal Powassan Encephalitis (Deer Tick Virus, Lineage II) in a Patient With Fever and Orchitis Receiving Rituximab.

Importance: Powassan virus is a rare but increasingly recognized cause of severe neurological disease. Objective: To highlight the diagnostic challenges and neuropathological findings in a fatal case of Powassan encephalitis caused by deer tick virus (lineage II) in a patient with follicular lymphoma receiving rituximab, with nonspecific anti-GAD65 antibodies, who was initially seen with fever and orchiepididymitis. Design, Setting, and Participants: Comparison of clinical, radiological, histological, and laboratory findings, including immunohistochemistry, real-time polymerase chain reaction, antibody detection, and unbiased sequencing assays, in a single case report (first seen in December 2016) at an academic medical center. Exposure: Infection with Powassan virus. Main Outcomes and Measures: Results of individual assays compared retrospectively. Results: In a 63-year-old man with fatal Powassan encephalitis, serum and cerebrospinal fluid IgM antibodies were not detected via standard methods, likely because of rituximab exposure. Neuropathological findings were extensive, including diffuse leptomeningeal and parenchymal lymphohistiocytic infiltration, microglial proliferation, marked neuronal loss, and white matter microinfarctions most severely involving the cerebellum, thalamus, and basal ganglia. Diagnosis was made after death by 3 independent methods, including demonstration of Powassan virus antigen in brain biopsy and autopsy tissue, detection of viral RNA in serum and cerebrospinal fluid by targeted real-time polymerase chain reaction, and detection of viral RNA in cerebrospinal fluid by unbiased sequencing. Extensive testing for other etiologies yielded negative results, including mumps virus owing to prodromal orchiepididymitis. Low-titer anti-GAD65 antibodies identified in serum, suggestive of limbic encephalitis, were not detected in cerebrospinal fluid. Conclusions and Relevance: Owing to the rarity of Powassan encephalitis, a high degree of suspicion is required to make the diagnosis, particularly in an immunocompromised patient, in whom antibody-based assays may be falsely negative. Unbiased sequencing assays have the potential to detect uncommon infectious agents and may prove useful in similar scenarios.

Delayed Diagnosis, Leprosy Reactions, and Nerve Injury Among Individuals With Hansen's Disease Seen at a United States Clinic.

Background. Hansen's disease (HD), or leprosy, is uncommon in the United States. We sought to describe the characteristics of patients with HD in a US clinic, including an assessment of delays in diagnosis and HD reactions, which have both been associated with nerve damage. Methods. A retrospective chart review was conducted on patients seen at an HD clinic in the southern United States between January 1, 2002 and January 31, 2014. Demographic and clinical characteristics were summarized, including delays in diagnosis, frequency of reactions, and other complications including peripheral neuropathy. Results. Thirty patients were seen during the study time period. The majority of patients were male (73%) and had multibacillary disease (70%). Brazil, Mexico, and the United States were the most frequent of the 14 countries of origin. Hansen's disease "reactions", severe inflammatory complications, were identified among 75% of patients, and nerve damage was present at diagnosis in 36% of patients. The median length of time between symptom onset and diagnosis was long at 12 months (range, 1-96), but no single factor was associated with a delay in diagnosis. Conclusions. The diagnosis of HD was frequently delayed among patients referred to our US clinic. The high frequency of reactions and neuropathy at diagnosis suggests that further efforts at timely diagnosis and management of this often unrecognized disease is needed to prevent the long-term sequelae associated with irreversible nerve damage.

Identification of Plasmodium falciparum specific translation inhibitors from the MMV Malaria Box using a high throughput in vitro translation screen.

BACKGROUND: A major goal in the search for new anti-malarial compounds is to identify new mechanisms of action or new molecular targets. While cell-based, growth inhibition-based screening have enjoyed tremendous success, an alternative approach is to specifically assay a given pathway or essential cellular process. METHODS: Here, this work describes the development of a plate-based, in vitro luciferase assay to probe for inhibitors specific to protein synthesis in Plasmodium falciparum through the use of an in vitro translation system derived from the parasite. RESULTS: Using the Medicines for Malaria Venture's Malaria Box as a pilot, 400 bioactive compounds with minimal human cytotoxicity profiles were screened, identifying eight compounds that displayed greater potency against the P. falciparum translation machinery relative to a mammalian translation system. Dose-response curves were determined in both translation systems to further characterize the top hit compound (MMV008270). CONCLUSIONS: This assay will be useful not only in future anti-malarial screening efforts but also in the investigation of P. falciparum protein synthesis and essential processes in P. falciparum biology.

Complex Type 2 Reactions in Three Patients with Hansen's Disease from a Southern United States Clinic.

In non-endemic countries, leprosy, or Hansen's disease (HD), remains rare and is often underrecognized. Consequently, the literature is currently lacking in clinical descriptions of leprosy complications in the United States. Immune-mediated inflammatory states known as reactions are common complications of HD. Type 1 reactions are typical of borderline cases and occur in 30% of patients and present as swelling and inflammation of existing skin lesions, neuritis, and nerve dysfunction. Type 2 reactions are systemic events that occur at the lepromatous end of the disease spectrum, and typical symptoms include fever, arthralgias, neuritis, and classic painful erythematous skin nodules known as erythema nodosum leprosum. We report three patients with lepromatous leprosy seen at a U.S. HD clinic with complicated type 2 reactions. The differences in presentations and clinical courses highlight the complexity of the disease and the need for increased awareness of unique manifestations of lepromatous leprosy in non-endemic areas.

Secondary Defense Chemicals in Milkweed Reduce Parasite Infection in Monarch Butterflies, Danaus plexippus.

In tri-trophic systems, herbivores may benefit from their host plants in fighting parasitic infections. Plants can provide parasite resistance in two contrasting ways: either directly, by interfering with the parasite, or indirectly, by increasing herbivore immunity or health. In monarch butterflies, the larval diet of milkweed strongly influences the fitness of a common protozoan parasite. Toxic secondary plant chemicals known as cardenolides correlate strongly with parasite resistance of the host, with greater cardenolide concentrations in the larval diet leading to lower parasite growth. However, milkweed cardenolides may covary with other indices of plant quality including nutrients, and a direct experimental link between cardenolides and parasite performance has not been established. To determine if the anti-parasitic activity of milkweeds is indeed due to secondary chemicals, as opposed to nutrition, we supplemented the diet of infected and uninfected monarch larvae with milkweed latex, which contains cardenolides but no nutrients. Across three experiments, increased dietary cardenolide concentrations reduced parasite growth in infected monarchs, which consequently had longer lifespans. However, uninfected monarchs showed no differences in lifespan across treatments, confirming that cardenolide-containing latex does not increase general health. Our results suggest that cardenolides are a driving force behind plant-derived resistance in this system.

Deciphering the glycogenome of schistosomes.

Schistosoma mansoni and other Schistosoma sp. are multicellular parasitic helminths (worms) that infect humans and mammals worldwide. Infection by these parasites, which results in developmental maturation and sexual differentiation of the worms over a period of 5-6 weeks, induces antibodies to glycan antigens expressed in surface and secreted glycoproteins and glycolipids. There is growing interest in defining these unusual parasite-synthesized glycan antigens and using them to understand immune responses, their roles in immunomodulation, and in using glycan antigens as potential vaccine targets. A key problem in this area, however, has been the lack of information about the enzymes involved in elaborating the complex repertoire of glycans represented by the schistosome glycome. Recent availability of the nuclear genome sequences for Schistosoma sp. has created the opportunity to define the glycogenome, which represents the specific genes and cognate enzymes that generate the glycome. Here we describe the current state of information in regard to the schistosome glycogenome and glycome and highlight the important classes of glycans and glycogenes that may be important in their generation.