ABSTRACT

Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in the numbers of extended-spectrum beta-lactamase-producing strains and the potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTI, create an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors FyuA, Hma, IreA, and IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys under conditions of challenge with UPEC strain CFT073 or strain 536. On the basis of these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use, including aluminum hydroxide gel (alum), monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA and dmLT-Hma displayed significant reductions in bladder colonization (86-fold and 32-fold, respectively), with 40% to 42% of mice having no detectable CFU. Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. On the basis of these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This report advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship.

IMPORTANCE Urinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli. Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface-exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.

ABSTRACT

DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification.

IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

This season’s flu vaccine was 45% effective overall and 55% effective among children and teens, the Centers for Disease Control and Prevention reported in February.

Health advocates across the country used National Public Health Week in April to highlight the important role of public health, which became even more crucial in the wake of the COVID-19 outbreak.

To the Editor—A 30-day injectable form of buprenorphine branded as SublocadeTM (Buprenorphine XR SQ) was approved by the FDA in 2017. This medication is administered by a health care professional subcutaneously in the abdomen to treat opioid use disorder. This long-acting delivery system holds great promise for many patients who have barriers to taking daily transmucosal buprenorphine-containing medications such as those with poor adherence to a daily medication. It is beneficial for those who have difficulty safely storing their medications, including patients who have children in the home, unstable housing, or live with others who have a use disorder. This product is also an option for patients who prefer mono-product buprenorphine. As Buprenorphine XR SQ is administered directly by a health care professional, it does not contain the abuse-deterrent naloxone that some patients feel causes side effects.

There are two ways to acquire Buprenorphine XR SQ: 1) order product from the distributor (buy and bill); or 2) dispensed from a specialty pharmacy for a specific patient (specialty pharmacy) [1]. For the buy and bill option, the health care setting must be certified through the Risk Evaluation and Mitigation Strategy (REMS) program and adhere to dispensing regulations [2]. We found this challenging to implement in the outpatient setting, thus we pursued the specialty pharmacy option. It ultimately took us nearly one year to complete the process.

The following are the barriers we faced with our first attempt. As a controlled substance, the medication must be stored in a refrigerated lockbox. Before...

With rising costs and below-average outcomes, North Carolina's health care value proposition is upside down. It's time for employers to lead transformative change.

Congenital myopathies are clinically and genetically heterogeneous, resulting from mutations in at least 30 different genes.¹ The classical presentation is neonatal hypotonia and nonprogressive weakness with normal creatine phosphokinase, although there is a broad range in terms of age at onset and clinical presentation. Historically, congenital myopathies have been defined and diagnosed based on muscle biopsy. However, with advances in genomics, genetics have taken primacy in the diagnostic pathway.²

In order to establish sustainable heat loading (heat removal and storage) in abandoned flooded mine workings it is important to understand the geomechanical impact of the cyclical heat loading caused by fluid injection and extraction. This is particularly important where significantly more thermal loading is planned than naturally occurs. A simple calculation shows that the sustainable geothermal heat flux from abandoned coal mines can provide less than a tenth of Scotland's annual domestic heating demand. Any heat removal greater than the natural heat flux will lead to heat mining unless heat storage options are also considered.

As a first step, a steady-state, fully saturated, 2D coupled hydromechanical model of a generalized section of pillar-and-stall workings has been created. Mine water rebound was modelled by increasing the hydrostatic pressure sequentially, in line with monitored mine water-level data from Midlothian, Scotland. The modelled uplift to water-level rise ratio of 1.4 mm m^–1 is of the same order of magnitude (1 mm m^–1) as that observed through interferometric synthetic aperture radar (InSAR) data in the coalfield due to mine water rebound. The modelled magnitude of shear stress at the pillar corners, as a result of horizontal and vertical displacement, is shown to increase linearly with water level. Mine heat systems are expected to cause smaller changes in pressure than those modelled but the results provide initial implications on the potential geomechanical impacts of mine water heat schemes which abstract or inject water and heat into pillar-and-stall coal mine workings.

Thematic collection: This article is part of the SJG Collection on Early-Career Research available at: https://www.lyellcollection.org/cc/SJG-early-career-research

Hydrogen sulfide (H₂S), a plant gasotransmitter, functions in the plant response to cadmium (Cd) stress, implying a role for cysteine desulfhydrase in producing H₂S in this process. Whether d-CYSTEINE DESULFHYDRASE (DCD) acts in the plant Cd response remains to be identified, and if it does, how DCD is regulated in this process is also unknown. Here, we report that DCD-mediated H₂S production enhances plant Cd tolerance in Arabidopsis (Arabidopsis thaliana). When subjected to Cd stress, a dcd mutant accumulated more Cd and reactive oxygen species and showed increased Cd sensitivity, whereas transgenic lines overexpressing DCD had decreased Cd and reactive oxygen species levels and were more tolerant to Cd stress compared with wild-type plants. Furthermore, the expression of DCD was stimulated by Cd stress, and this up-regulation was mediated by a Cd-induced transcription factor, WRKY13, which bound to the DCD promoter. Consistently, the higher Cd sensitivity of the wrky13-3 mutant was rescued by the overexpression of DCD. Together, our results demonstrate that Cd-induced WRKY13 activates DCD expression to increase the production of H₂S, leading to higher Cd tolerance in plants.

Plants have evolved complex physiological and biochemical mechanisms to adapt to a heterogeneous soil phosphorus environment. PHOSPHATE2 (PHO2) is a phosphate (Pi) starvation-signaling regulator involved in maintaining Pi homeostasis in plants. Arabidopsis (Arabidopsis thaliana) PHO2 targets PHOSPHATE TRANSPORTER1 (PHT1) and PHO1 for degradation, whereas rice (Oryza sativa) PHO2 is thought to mediate PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 degradation. However, it is unclear whether and how PHO2 is post-translationally regulated. Here, we show that in rice, the CASEIN KINASE2 (OsCK2) catalytic subunit OsCK2α3 interacts with OsPHO2 in vitro and in vivo in vascular tissues cells, and phosphorylates OsPHO2 at Ser-841. Phosphorylated OsPHO2 is degraded more rapidly than native OsPHO2 in cell-free degradation assays. OsPHO2 interacts with OsPHO1 and targets it for degradation through a multivesicular body-mediated pathway. PHO1 mutation partially rescued the pho2 mutant phenotype. Further genetic analysis showed that a nonphosphorylatable version of OsPHO2 rescued the Ospho2 phenotype of high Pi accumulation in leaves better than native OsPHO2. In addition to the previously established role of OsCK2 in negatively regulating endoplasmic reticulum exit of PHT1 phosphate transporters, this work uncovers a role for OsCK2α3 in modulating Pi homeostasis through regulating the phosphorylation status and abundance of OsPHO2 in rice.

Phosphatidylcholine and phosphatidylethanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or ethanolamine (Etn) catalyzed by either choline or ethanolamine kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn kinases and Cho kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.

The coronavirus disease 2019 (COVID-19) epidemic, which was first reported in December 2019 in Wuhan, China, has caused 80 904 confirmed cases as of 9 March 2020, with 28 673 cases being reported outside of China. It has been declared a pandemic by the World Health Organization (WHO), has exhibited human-to-human transmissibility and has spread rapidly across countries [1]. Although the Chinese government has taken various measures to control city-to-city transmission (e.g. shutting down cities, extending holidays) and many other countries have implemented measures (such as airport screening and testing patients who have reported symptoms), the number of cases is still increasing quickly throughout the world.

The World Health Organization (WHO) has listed moxifloxacin and linezolid among the preferred "group A" drugs in the treatment of multidrug-resistant (MDR)-tuberculosis (TB) [1]. Therapeutic drug monitoring (TDM) could potentially optimise MDR-TB therapy, since moxifloxacin and linezolid show large pharmacokinetic variability [1–4]. TDM of moxifloxacin focuses on identifying patients with low drug exposure who are at risk of treatment failure and acquired fluoroquinolone resistance [5, 6]. Alternatively, TDM of linezolid strives to reduce toxicity while ensuring an adequate drug exposure because of its narrow therapeutic index [1, 3, 7].

Objective

To investigate the frequency of levodopa-induced dyskinesia in dementia with Lewy bodies (DLBs) and Parkinson disease with dementia (PDD) and compare these frequencies with patients with incident Parkinson disease (PD) through a population-based cohort study.

Objective

To use the variations in neurology consultations requested by emergency department (ED) physicians to identify opportunities to implement multidisciplinary interventions in an effort to reduce ED overcrowding.

Background

Transient loss of consciousness (TLOC) is a common reason for presentation to primary/emergency care; over 90% are because of epilepsy, syncope, or psychogenic non-epileptic seizures (PNES). Misdiagnoses are common, and there are currently no validated decision rules to aid diagnosis and management. We seek to explore the utility of machine-learning techniques to develop a short diagnostic instrument by extracting features with optimal discriminatory values from responses to detailed questionnaires about TLOC manifestations and comorbidities (86 questions to patients, 31 to TLOC witnesses).

In the current era of technological advances in medicine, public interest seems focused on advances in laboratory testing, imaging, and surgical instrumentation. Modern-day expectations in the diagnostic part of medicine appear to demand answers which are instant, specific, and without ambiguity. This is consistent with the idea of developing the science of medicine. However, many still consider obtaining a diagnosis through a careful and thoughtful history as an example of the art of medicine.

Cystinuria is an autosomal recessive disorder characterized by excessive urinary excretion of cystine, resulting in recurrent cystine kidney stones, often presenting in childhood. Current treatment options for cystinuria include dietary and/or fluid measures and potassium citrate to reduce cystine excretion and/or increase solubility. Tiopronin and D-penicillamine are used in refractory cases to bind cystine in urine, albeit with serious side effects. A recent study revealed efficacy of nutritional supplement α-lipoic acid (ALA) treatment in preventing kidney stones in a mouse model of cystinuria. Here, we report 2 pediatric patients (6 and 15 years old) with cystinuria who received regular doses of ALA in addition to conventional therapy with potassium citrate. Both patients tolerated ALA without any adverse effects and had reduced frequency of symptomatic and asymptomatic kidney stones with disappearance of existing kidney stones in 1 patient after 2 months of ALA therapy. ALA treatment markedly improved laboratory markers of cystine solubility in urine with increased cystine capacity (–223 to –1 mg/L in patient 1 and +140 to +272 mg/L in patient 2) and decreased cystine supersaturation (1.7 to 0.88 in patient 1 and 0.64 to 0.48 in patient 2) without any changes in cystine excretion or urine pH. Our findings suggest that ALA improves solubility of cystine in urine and prevents stone formation in patients with cystinuria who do not respond to diet and citrate therapy.

Systemic sclerosis sine scleroderma (ssSSc) is a rare variant of systemic sclerosis, with only one pediatric case reported in the medical literature to date. Pulmonary arterial hypertension as the presenting feature of ssSSc is extremely rare, even in adults, and so far has never been reported in children. We report, for the first time, a case of pediatric ssSSc in a 3-year-old girl, who presented with interstitial lung disease and pulmonary hypertension. The patient was prescribed early aggressive pulmonary vasodilators combined with anti-inflammatory medications. The clinical response was good, and her current condition at 12 years of age is remarkable, considering the high mortality rates reported in adults. We underscore the importance of early aggressive treatment in future cases of similar presentation.

BACKGROUND AND OBJECTIVES:

Early diagnosis of cerebral palsy (CP) is critical in obtaining evidence-based interventions when plasticity is greatest. In 2017, international guidelines for early detection of CP were published on the basis of a systematic review of evidence. Our study aim was to reduce the age at CP diagnosis throughout a network of 5 diverse US high-risk infant follow-up programs through consistent implementation of these guidelines.

In plant–pathogen relations, disease symptoms arise from the interaction of the host and pathogen genomes. Host–pathogen functional gene interactions are well described, whereas little is known about how the pathogen genetic variation modulates both organisms’ transcriptomes. To model and generate hypotheses on a generalist pathogen control of gene expression regulation, we used the Arabidopsis thaliana–Botrytis cinerea pathosystem and the genetic diversity of a collection of 96 B. cinerea isolates. We performed expression-based genome-wide association (eGWA) for each of 23,947 measurable transcripts in Arabidopsis (host), and 9267 measurable transcripts in B. cinerea (pathogen). Unlike other eGWA studies, we detected a relative absence of locally acting expression quantitative trait loci (cis-eQTL), partly caused by structural variants and allelic heterogeneity hindering their identification. This study identified several distantly acting trans-eQTL linked to eQTL hotspots dispersed across Botrytis genome that altered only Botrytis transcripts, only Arabidopsis transcripts, or transcripts from both species. Gene membership in the trans-eQTL hotspots suggests links between gene expression regulation and both known and novel virulence mechanisms in this pathosystem. Genes annotated to these hotspots provide potential targets for blocking manipulation of the host response by this ubiquitous generalist necrotrophic pathogen.

There is increasing interest in developing diagnostics that discriminate individual mutagenic mechanisms in a range of applications that include identifying population-specific mutagenesis and resolving distinct mutation signatures in cancer samples. Analyses for these applications assume that mutagenic mechanisms have a distinct relationship with neighboring bases that allows them to be distinguished. Direct support for this assumption is limited to a small number of simple cases, e.g., CpG hypermutability. We have evaluated whether the mechanistic origin of a point mutation can be resolved using only sequence context for a more complicated case. We contrasted single nucleotide variants originating from the multitude of mutagenic processes that normally operate in the mouse germline with those induced by the potent mutagen N-ethyl-N-nitrosourea (ENU). The considerable overlap in the mutation spectra of these two samples make this a challenging problem. Employing a new, robust log-linear modeling method, we demonstrate that neighboring bases contain information regarding point mutation direction that differs between the ENU-induced and spontaneous mutation variant classes. A logistic regression classifier exhibited strong performance at discriminating between the different mutation classes. Concordance between the feature set of the best classifier and information content analyses suggest our results can be generalized to other mutation classification problems. We conclude that machine learning can be used to build a practical classification tool to identify the mutation mechanism for individual genetic variants. Software implementing our approach is freely available under an open-source license.

Key Points

Key Points

Key Points

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 x 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 x 10³ CFU (50 LD₅₀) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Brucella spp. are facultative intracellular bacteria notorious for their ability to induce a chronic, and often lifelong, infection known as brucellosis. To date, no licensed vaccine exists for prevention of human disease, and mechanisms underlying chronic illness and immune evasion remain elusive. We and others have observed that B cell-deficient mice challenged with Brucella display reduced bacterial burden following infection, but the underlying mechanism has not been clearly defined. Here, we show that at 1 month postinfection, B cell deficiency alone enhanced resistance to splenic infection ~100-fold; however, combined B and T cell deficiency did not impact bacterial burden, indicating that B cells only enhance susceptibility to infection when T cells are present. Therefore, we investigated whether B cells inhibit T cell-mediated protection against Brucella. Using B and T cell-deficient Rag1^–/– animals as recipients, we demonstrate that adoptive transfer of CD4⁺ T cells alone confers marked protection against Brucella melitensis that is abrogated by cotransfer of B cells. Interestingly, depletion of CD4⁺ T cells from B cell-deficient, but not wild-type, mice enhanced susceptibility to infection, further confirming that CD4⁺ T cell-mediated immunity against Brucella is inhibited by B cells. In addition, we found that the ability of B cells to suppress CD4⁺ T cell-mediated immunity and modulate CD4⁺ T cell effector responses during infection was major histocompatibility complex class II (MHCII)-dependent. Collectively, these findings indicate that B cells modulate CD4⁺ T cell function through an MHCII-dependent mechanism which enhances susceptibility to Brucella infection.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.

IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the "core domain" and the N-terminal "arm domain." The "arm domain" contains the residues from M1 to N83, and the "core domain" contains the residues from N84 to A273. A structure analysis reveals that the "core domain" shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the "arm domain" is unique to ASFV. Further, experiments indicated that the "arm domain" plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen.

IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique "arm domain" has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

Human adenoviruses have many attractive features for gene therapy applications. However, the high prevalence of preexisting immunity against these viruses in general populations worldwide has greatly limited their clinical utility. In addition, the most commonly used human adenovirus, human adenovirus subgroup C serotype 5 (HAd5), when systemically administered, triggers systemic inflammation and toxicity, with the liver being the most severely affected organ. Here, we evaluated the utility and safety of a new low-seroprevalence gorilla adenovirus (GAd; GC46) as a gene transfer vector in mice. Biodistribution studies revealed that systemically administered GAd had a selective and robust lung endothelial cell (EC) tropism with minimal vector expression throughout many other organs and tissues. Administration of a high dose of GAd accomplished extensive transgene expression in the lung yet elicited no detectable inflammatory histopathology in this organ. Furthermore, GAd, unlike HAd5, did not exhibit hepatotropism or induce liver inflammatory toxicity in mice, demonstrating the exceptional safety profile of the vector vis-à-vis systemic utility. We further demonstrated that the GAd capsid fiber shared the flexibility of the HAd5 equivalent for permitting genetic modification; GAd with the pan-EC-targeting ligand myeloid cell-binding peptide (MBP) incorporated in the capsid displayed a reduced lung tropism and efficiently retargeted gene expression to vascular beds in other organs.

IMPORTANCE In the aggregate, our mouse studies suggest that GAd is a promising gene therapy vector that utilizes lung ECs as a source of therapeutic payload production and a highly desirable toxicity profile. Further genetic engineering of the GAd capsid holds the promise of in vivo vector tropism modification and targeting.

The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4⁺ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4⁺ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4⁺ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought.

IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4⁺ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4⁺ T cells and the FDC reservoir in vitro. Although CAR-T cells eliminated CD4⁺ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)₃] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.

IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.

Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.

IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species.

IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.

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