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While mRNA vaccines have been effective in combating SARS-CoV-2, waning of vaccine-induced antibody responses and lack of vaccine-induced respiratory tract immunity contribute to ongoing infection and transmission. In this work, we compare and contrast intranasal (i.n.) and intramuscular (i.m.) administration of a SARS-CoV-2 self-amplifying RNA (saRNA) vaccine delivered by a nanostructured lipid carrier (NLC). Both i.m. and i.n. vaccines induce potent systemic serum neutralizing antibodies, bone marrow-resident IgG-secreting cells, and robust lymphoid tissue T cell immune responses. The i.n. vaccine additionally induces robust respiratory mucosal immune responses, including SARS-CoV-2-reactive lung-resident memory and lung-homing T cell populations. As a booster following previous i.m. vaccination, the i.n. vaccine also elicits the development of mucosal virus-specific T cells. Both the i.m. and i.n. administered vaccines protect hamsters from infection-associated morbidity upon viral challenge, significantly reducing viral loads and preventing challenged hamsters from transmitting virus to naive cagemates. This saRNA vaccine’s potent systemic immunogenicity, and additional mucosal immunogenicity when delivered i.n., may be key for combating SARS-CoV-2 and other respiratory pathogens.One Sentence SummaryIntranasal SARS-CoV-2 saRNA vaccination induces systemic and mucosal immunity in mice, and prevents morbidity and blocks viral transmission in hamsters.

CD8+ T cell responses to pulmonary challenges are primed by lung migratory dendritic cells (mDCs), which capture antigens in the lungs and migrate to the lung-draining mediastinal lymph node (med-L...

CD8(+) T cell responses to pulmonary challenges are primed by lung migratory dendritic cells (mDCs), which capture antigens in the lungs and migrate to the lung-draining mediastinal lymph node (med-LN) to activate T cells. Notably, the lungs and the spleen are not connected by the lymphatic vasculature. Thus, the current paradigm suggests that in response to respiratory virus infections that are restricted to the respiratory tract, priming of T cell responses by lung mDCs takes place entirely in the med-LN. Our results challenge this “LN-centric” paradigm by demonstrating that during influenza virus infection, lung mDCs egress the med-LN and traffic to the spleen, where they prime influenza-specific CD8(+) T cells. Importantly, CD8(+) T cells primed in the spleen are transcriptionally distinct and have enhanced ability to differentiate into long-lived memory cells compared to med-LN-primed counterparts. Thus, our data identify a lung mDC trafficking pathway that connects the lungs with the spleen.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

NAD, a key co-enzyme required for cell metabolism, is synthesized via two pathways in most organisms. Since schistosomes apparently lack enzymes required for de novo NAD biosynthesis, we evaluated whether these parasites, which infect >200 million people worldwide, maintain NAD homeostasis via the NAD salvage biosynthetic pathway. We found that intracellular NAD levels decline in schistosomes treated with drugs that block production of nicotinamide or nicotinamide mononucleotide–known NAD precursors in the non-deamidating salvage pathway. Moreover, in vitro inhibition of the NAD salvage pathway in schistosomes impaired egg production, disrupted the outer membranes of both immature and mature parasites and caused loss of mobility and death. Inhibiting the NAD salvage pathway in schistosome-infected mice significantly decreased NAD levels in adult parasites, which correlated with reduced egg production, fewer liver granulomas and parasite death. Thus, schistosomes, unlike their mammalian hosts, appear limited to one metabolic pathway to maintain NAD-dependent metabolic processes., Author summary Schistosomiasis (snail fever) is a deadly parasitic disease that affects more than 200 million people worldwide and, if not treated, can lead to death. This disease is caused by parasitic worms called schistosomes that feed on the host blood and lay hundreds of eggs each day that damage the liver and kidneys. Therapies to treat schistosomiasis are limited. The most widely-used anti-schistosomal drug, praziquantel, is not effective against immature parasites and adult worms can, in some cases, become resistant to this drug. It is therefore important to find new therapies to treat this deadly disease. In this study, we observed that schistosomes cannot use amino acids to make Nicotinamide Adenine Dinucleotide (NAD)–a key cellular metabolite found in all living organisms. Instead, these parasites salvage NAD by scavenging vitamins from the host. We observed that disruption of this NAD salvage pathway negatively impacts metabolism, reproduction and survival of both adult and immature worms. As such, targeting the parasite’s NAD salvage pathway is a promising therapeutic approach for the treatment of snail fever.

Memory B cells are found in lymphoid and non-lymphoid tissues, suggesting that some may be tissue-resident cells. Here we show that pulmonary influenza infection elicited lung-resident memory B cells (BRM cells) that were phenotypically and functionally distinct from their systemic counterparts. BRM cells were established in the lung early after infection, in part because their placement required local antigen encounter. Lung BRM cells, but not systemic memory B cells, contributed to early plasmablast responses following challenge infection. Following secondary infection, antigen-specific BRM cells differentiated in situ, whereas antigen-non-specific BRM cells were maintained as memory cells. These data demonstrate that BRM cells are an important component of immunity to respiratory viruses such as influenza virus and suggest that vaccines designed to elicit BRM cells must deliver antigen to the lungs.

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

The Dolomite Alps of northeastern Italy experience debris flows with great frequency during the summer months. An ample supply of unconsolidated material on steep slopes and a summer season climate regime characterized by recurrent thunderstorms combine to produce an abundance of these destructive hydrogeologic events. In the past debris flow events have been studied primarily in the context of their geologic and geomorphic characteristics. The atmospheric contribution to these mass wasting events has been limited to recording rainfall and developing intensity thresholds for debris mobilization. This study aims to expand the examination of atmospheric processes that preceded both locally intense convective rainfall (LICR) and debris flows in the Dolomite region. 500 hPa pressure level plots of geopotential heights were constructed for a period of three days prior to debris flow events to gain insight into the synoptic scale processes which provide an environment conducive to LICR in the Dolomites. Cloud-to-ground (CG) lightning flash data recorded at the meso-scale were incorporated to assess the convective environment proximal to debris flow source regions. Twelve events were analyzed and from this analysis three common synoptic scale circulation patterns were identified. Evaluation of CG flashes at smaller spatial and temporal scales illustrated that convective processes vary in their production of CG flashes (total number) and the spatial distribution of flashes can also be quite different between events over longer periods. During the 60 min interval immediately preceding debris flow a majority of cases exhibited spatial and temporal collocation of LICR and CG flashes. Also a number of CG flash parameters were found to be significantly correlated to rainfall intensity prior to debris flow initiation.

SmNACE is a NAD catabolizing enzyme expressed on the outer tegument of S. mansoni, a human parasite that is one of the major agents of the neglected tropical disease schistosomiasis. Recently, we identified aroylhydrazone derivatives capable of inhibiting the recombinant form of the enzyme with variable potency (IC50 ranging from 88 μM to 33 nM). In the present study, we investigated the mechanism of action of the least potent micromolar inhibitor (compound 1) and the most potent nanomolar inhibitor (compound 2) in the series on both the recombinant and native SmNACE enzymes. Using mass spectroscopy, spectrophotometry, and activity assays under different experimental conditions, we demonstrated that the >3 log gain in potency against recombinant SmNACE by this class of compounds is dependent on the formation of a coordination complex with metal cations, such as Ni(II), Zn(II), and Fe(II), that are loaded on the protein surface. Testing the compounds on live parasites, we observed that only the weak microm...

Currently, no uniform method exists for determining the optimal areal unit to analyze National Lightning Detection Network (NLDN) data. To address this problem, this paper utilizes the capabilities of modern geographic information systems (GIS) software to develop a consistent method for identifying areal analysis units while considering the location accuracy of the NLDN. Five grid cells were created at spatial resolutions of 0.1°, 0.2°, 0.3°, 0.4°, and 0.5°. To create cloud-to-ground (CG) lightning strikes, random points were generated at nine densities ranging from 1 to 9 strikes per square kilometer. A buffer of 500 m was placed around each random point to account for the margin of error in NLDN location accuracy. Random points that, when buffered to 500 m, still remained completely within the study region were evaluated as a percentage of all of the strikes to determine accuracy. The greatest accuracy of 95.88% was observed in the 0.5° grid cell at a density of 9 strikes per square kilometer. The lowest accuracy of 80.59% occurred in the 0.1° grid cell at a density of 4 strikes per square kilometer. There was little variation between the accuracies in similar grid cells regardless of their density, suggesting that CG flash density will have little effect on accuracy when selecting spatial resolution. Gains in accuracy diminished as spatial resolution increased. Gains in accuracy between the 0.1° and the 0.2° grid cells are approximately 9%. Gains in accuracy between the 0.4° and the 0.5° grid cells are less than 1%. To achieve 95% accuracy, a spatial resolution of no less than 0.4° is required.

Flash flood and debris flow reports from Storm Data and the U.S. Geological Survey (USGS) are used to investigate the relationship between hazardous hydrological responses, convective rainfall, and cloud-to-ground (CG) lightning flash parameters. Basins burned by the Coal Seam and Missionary Ridge wildfires during the summer of 2002 in western Colorado were selected as primary study areas. The North American monsoon (NAM) air mass played a pivotal role in providing low-level moisture over much of Colorado during each of the 12 hydrological events identified. Surface θe values as high as 354 K were calculated over western Colorado in a composite analysis that also saw a θe ridge through 500 hPa extending northward into Nebraska and southern South Dakota. Storm-total CG flashes were as high as 718, and the median flash total for the population of events was 256. Mean 5-min CG flash intensity for the events was 18.1 flashes. The mean rainfall intensity associated with the 12 hydrological events was ...

Some memory CD8+ T cells are programmed during the contraction phase of the primary response to have robust secondary responsiveness, a phenomenon termed memory programming. However, the underlying mechanism of the memory programming is not entirely understood. Using an influenza virus infection model, we show here that memory CD8+ T cell responses to the more abundant nucleoprotein (NP) antigen more vigorously proliferate than CD8+ T cell responses to the less abundant polymerase (PA) antigen. Interestingly, in spite of their differential abilities to mount secondary responses, memory NP and PA-specific CD8+ T cells are present in comparable frequencies in the lung parenchyma and airways during the memory phase. However, whereas NP-specific CD8+ T cells express low levels of CD103 and CD69, PA-specific CD8+ T cells are predominately CD103hiCD69hi, a phenotype characteristic of tissue resident memory (Trm) populations. Importantly, our data indicate that persistent antigen presentation during the contraction phase of the primary response prevented the differentiation of Trm cells. As a result, whereas prolonged antigen presentation by CD103− CD11bhi dendritic cells (DCs) during the late phase of the primary response programmed NP-specific CD8+ T cells to potently expand after re-challenge, it prevented the differentiation of NP-specific Trm cells. Collectively, our results demonstrate that CD8+ T cell responses to less abundant antigens, such as PA, preferentially develop into tissue resident memory populations.

By means of a phenomenon termed “the Warburg effect,” tumor cells shift their energy production by mitochondrial oxidative phosphorylation to aerobic glycolysis, resulting in the upregulation of glucose consumption and increased cellular oxidative and nitrosative stress. To compensate for such toxic levels of ROS/RNS, cancer cells rely heavily on their antioxidant defense mechanisms, which are largely controlled by the NAD(P)/NAD(P)H redox partners. We found that the modulation of NAD metabolism in vivo, specifically via the deletion of the NAD glycohydrolase CD38, resulted in increased production of intrinsic ROS and increased DNA damage following exposure to chemotherapeutics. Furthermore, in vitro experiments showed that CD38 knockdown in CD38-expressing tumor cells prevented the generation of stable transfectants, highlighting a role for CD38 in tumor cell survival. In light of these findings, we hypothesized that pharmacological inhibition of CD38 may be an effective therapy for the treatment of hematological cancers, in particular those which uniformly overexpress CD38, such as MM and chronic lymphocytic leukemia. Indeed, treatment of human MM cell lines LP-1 and KMS-12-PE with CD38 antagonists sensitized the cells to standard ROS-inducing chemotherapeutics. We conducted a high-throughput screening (HTS) campaign of over two hundred thousand unique and non-proprietary lead-like compounds using an optimized and miniaturized HTS based on a luminescent NAD quantitation platform. Five hundred active hits were analyzed for toxicity using a cell-based HTS assay purposely designed with CD38-negative HEK293 cells to avoid elimination of desirable compounds toxic to CD38-positive cells. Hits with non-specific properties, such as PAINS (Pan Assay Interference Compounds), were removed by computational filtering, and the remaining compounds were tested for inhibition of human CD38 activity in cells. The last phase of the compound progression pathway involved testing for non-selective inhibition of other NAD-consuming enzymes, namely Poly(ADP-ribose) polymerase-1, and Sirtuin-1, which led to the identification of two distinct chemical series that exhibit >10-fold selectivity for human CD38. Hit-to-lead chemistry is currently underway to synthesize key analogs by rational drug design. In summary, our data suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing hematological cancers. Our HTS campaign efforts are paving the way for the discovery and development of potent and selective small-molecule inhibitors of CD38. Citation Format: Davide Botta, Tulin Dadali, Betty J. Mousseau, Fen Zhou, Michael D. Schultz, Esther Zumaquero, Anna Manouvakhova, Melinda I. Sosa, Sara N. McKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E. Lucille White, Indira Padmalayam, Wei Zhang, Maaike Everts, Corinne E. Augelli-Szafran, James R. Bostwick, Mark J. Suto, Frances E. Lund. High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-055.

From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease., Author Summary Several historical epidemic waves of plague have been attributed to Yersinia pestis, the etiologic agent of modern plague. The most famous of these was the second pandemic which was active in Europe from AD 1347 until 1750, and began with the ‘Black Death’. The most informative method to establish the etiological nature of these ancient infections should be the analysis of ancient DNA, but the results of this method have been controversial. Here, by combining ancient DNA analyses and protein-specific detection, we demonstrate unambiguously that Y. pestis caused the Black Death. Furthermore, we show that at least two variants of Y. pestis spread over Europe during the second pandemic. The analysis of up to 20 diagnostic markers reveals that the two variants evolved near the time that phylogenetic branches 1 and 2 separated and may no longer exist. Our results thus resolve a long-standing debate about the etiology of the Black Death and provide key information about the evolution of the plague bacillus and the spread of the disease during the Middle Ages.

[1] Cloud-to-ground (CG) lightning flash parameters collected by the National Lightning Detection Network were analyzed in conjunction with rainfall observations near Mitchell Creek (MC) at the Coal Seam Wildfire site in western Colorado, USA. Nine thunderstorms produced significant CG flashes in the area surrounding MC from 28 June (fire containment) to 5 August 2002. A debris flow was generated at MC by one of these storms at ∼2058 LT on 5 August 2002. This study compares the CG flash parameters and rainfall characteristics of the 5 August thunderstorm with the eight thunderstorms (control group) that did not produce a hazardous hydrologic response at MC. The CG flash patterns and a synoptic analysis suggest that the 5 August thunderstorms occurred during a North American Monsoon “burst” period with a strong southwesterly surge of moisture advecting as far north as central Wyoming. The 5 August thunderstorm sequence was bimodal and more intense in terms of CG flash totals and rainfall rates than the control group. Moist southwesterly flow at lower levels and dry southeasterly flow in the upper troposphere may have enhanced orographically forced convection during the evening of 5 August. CG flashes and rainfall at spatial scales of 100 × 100 km, 50 × 50 km, and 25 × 25 km around MC confirm that the 5 August episode was more intense than any of the control group. The analysis concluded that the time of first flash and the number of consecutive 5-min intervals with CG flashes were temporally related with intense rainfall at the debris flow site. Additionally, there was a strong correlation between flash clusters within 40 km of MC and rainfall intensity at the site. The results of this study suggest that CG flash parameters may prove beneficial in modeling rainfall intensity thresholds in areas burned by wildfire.