A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV-2 challenge in rhesus macaques

BackgroundPersistent transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has given rise to a COVID-19 pandemic. Several vaccines, evoking protective spike antibody responses, conceived in 2020, are being deployed in mass public health vaccination programs. Recent data suggests, however, that as sequence variation in the spike genome accumulates, some vaccines may lose efficacy. MethodsUsing a macaque model of SARS-CoV-2 infection, we tested the efficacy of a peptide-based vaccine targeting MHC Class I epitopes on the SARS-CoV-2 nucleocapsid protein. We administered biodegradable microspheres with synthetic peptides and adjuvants to rhesus macaques. Unvaccinated control and vaccinated macaques were challenged with 1 x 108 TCID50 units of SARS-CoV-2, followed by assessment of clinical symptoms, viral load, chest radiographs, sampling of peripheral blood and bronchoalveolar lavage (BAL) fluid for downstream analysis. ResultsVaccinated animals were free of pneumonia-like infiltrates characteristic of SARS-CoV-2 infection and presented with lower viral loads relative to controls. Gene expression in cells collected from BAL samples of vaccinated macaques revealed a unique signature associated with enhanced development of adaptive immune responses relative to control macaques. ConclusionsWe demonstrate that a room temperature stable peptide vaccine based on known immunogenic HLA Class I bound CTL epitopes from the nucleocapsid protein can provide protection against SARS-CoV-2 infection in non-human primates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/441228v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@c0730aorg.highwire.dtl.DTLVardef@c0bff7org.highwire.dtl.DTLVardef@7b1ea6org.highwire.dtl.DTLVardef@11950a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dysregulation of genes and microRNAs in localized aggressive periodontitis.

AIM: Previous data from our laboratory have demonstrated that localized aggressive periodontitis (LAP) patients produce elevated levels of pro-inflammatory cytokines in response to TLR4 and TLR2 ligation compared to unrelated and periodontally healthy controls (HC). The aim of the present work is to evaluate the contribution of TLR-related gene expression and miRNA regulation in LAP disease. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMCs) from LAP and health control (HC) patients were isolated. Gene and miRNA expression involved in TLR signalling pathway and immunopathology were evaluated in unstimulated PBMCs by real-time PCR (RT-PCR). RESULTS: TICAM-1 (TRIF), FOS, IRAK1, TLR2 and CCL2 genes and the miRNAs miR-9-5p, miR-155-5p and 203a-3p, miR-147a, miR-182-5p and miR-183-5p were significantly up-regulated in LAP compared to HC. CONCLUSIONS: Most of the genes and miRNAs overexpressed here are directly or indirectly related to immune response and inflammation. This profile supports our previous findings that suggests LAP patients have a "hyper-responsive" phenotype upon activation of TLR pathway by periodontal pathogens.

Flash and grab: deep-diving southern elephant seals trigger anti-predator flashes in bioluminescent prey.

Bioluminescence, which occurs in approximately 80% of the world's mesopelagic fauna, can take the form of a low-intensity continuous glow (e.g. for counter-illumination or signalling) or fast repetitions of brighter anti-predatory flashes. The southern elephant seal (SES) is a major consumer of mesopelagic organisms, in particular the abundant myctophid fish, yet the fine-scale relationship between this predator's foraging behaviour and bioluminescent prey remains poorly understood. We hypothesised that brief, intense light emissions should be closely connected with prey strikes when the seal is targeting bioluminescent prey that reacts by emitting anti-predator flashes. To test this, we developed a biologging device containing a fast-sampling light sensor together with location and movement sensors to measure simultaneously anti-predator bioluminescent emissions and the predator's attack motions with a 20 ms resolution. Tags were deployed on female SES breeding at Kerguelen Islands and Península Valdés, Argentina. In situ light levels in combination with duration of prey capture attempts indicated that seals were targeting a variety of prey types. For some individuals, bioluminescent flashes occurred in a large proportion of prey strikes, with the timing of flashes closely connected with the predator's attack motion, suggestive of anti-predator emissions. Marked differences across individuals and location indicate that SES do exploit bioluminescent organisms but the proportion of these in the diet varies widely with location. The combination of wideband light and acceleration data provides new insight into where and when different prey types are encountered and how effectively they might be captured.

An Effective CTL Peptide Vaccine for Ebola Zaire Based on Survivors' CD8+ Targeting of a Particular Nucleocapsid Protein Epitope with Potential Implications for COVID-19 Vaccine Design

The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus.

Blue-Enriched Lighting for Older People Living in Care Homes: Effect on Activity, Actigraphic Sleep, Mood and Alertness.

OBJECTIVE: Environmental (little outdoor light; low indoor lighting) and age-related physiological factors (reduced light transmission through the ocular lens, reduced mobility) contribute to a light-deprived environment for older people living in care homes. METHODS: This study investigates the effect of increasing indoor light levels with blue-enriched white lighting on objective (rest-activity rhythms, performance) and self-reported (mood, sleep, alertness) measures in older people. Eighty residents (69 female), aged 86 ± 8 yrs (mean ± SD), participated (MMSE 19 ± 6). Overhead fluorescent lighting was installed in communal rooms (n=20) of seven care homes. Four weeks of blue-enriched white lighting (17000 K ≅ 900 lux) were compared with four weeks of control white lighting (4000 K ≅ 200 lux), separated by three weeks wash-out. Participants completed validated mood and sleep questionnaires, psychomotor vigilance task (PVT) and wore activity and light monitors (AWL). Rest-activity rhythms were assessed by cosinor, non-parametric circadian rhythm (NPCRA) and actigraphic sleep analysis. Blue-enriched (17000 K) light increased wake time and activity during sleep decreasing actual sleep time, sleep percentage and sleep efficiency (p < 0.05) (actigraphic sleep). Compared to 4000 K lighting, blue-enriched 17000 K lighting significantly (p < 0.05) advanced the timing of participants' rest-activity rhythm (cosinor), increased daytime and night-time activity (NPCRA), reduced subjective anxiety (HADA) and sleep quality (PSQI). There was no difference between the two light conditions in daytime alertness and performance (PVT). CONCLUSION: Blue-enriched lighting produced some positive (increased daytime activity, reduced anxiety) and negative (increased night-time activity, reduced sleep efficiency and quality) effects in older people.

Sperm whales reduce foraging effort during exposure to 1-2 kHz sonar and killer whale sounds.

The time and energetic costs of behavioral responses to incidental and experimental sonar exposures, as well as control stimuli, were quantified using hidden state analysis of time series of acoustic and movement data recorded by tags (DTAG) attached to 12 sperm whales (Physeter macrocephalus) using suction cups. Behavioral state transition modeling showed that tagged whales switched to a non-foraging, non-resting state during both experimental transmissions of low-frequency active sonar from an approaching vessel (LFAS; 1-2 kHz, source level 214 dB re 1 µPa m, four tag records) and playbacks of potential predator (killer whale, Orcinus orca) sounds broadcast at naturally occurring sound levels as a positive control from a drifting boat (five tag records). Time spent in foraging states and the probability of prey capture attempts were reduced during these two types of exposures with little change in overall locomotion activity, suggesting an effect on energy intake with no immediate compensation. Whales switched to the active non-foraging state over received sound pressure levels of 131-165 dB re 1 µPa during LFAS exposure. In contrast, no changes in foraging behavior were detected in response to experimental negative controls (no-sonar ship approach or noise control playback) or to experimental medium-frequency active sonar exposures (MFAS; 6-7 kHz, source level 199 re 1 µPa m, received sound pressure level [SPL] = 73-158 dB re 1 µPa). Similarly, there was no reduction in foraging effort for three whales exposed to incidental, unidentified 4.7-5.1 kHz sonar signals received at lower levels (SPL = 89-133 dB re 1 µPa). These results demonstrate that similar to predation risk, exposure to sonar can affect functional behaviors, and indicate that increased perception of risk with higher source level or lower frequency may modulate how sperm whales respond to anthropogenic sound.

Quantitative analysis of three-dimensional human mammary epithelial tissue architecture reveals a role for tenascin-C in regulating c-met function.

Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.

Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway.

Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and beta-catenin were required for expression of Pdgfralpha and Pdgfrbeta and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfralpha and Pdgfrbeta as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfralpha/beta expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung.

Inflammation, growth factors, and pulmonary vascular remodeling.

Inflammatory processes are prominent in various types of human and experimental pulmonary hypertension (PH) and are increasingly recognized as major pathogenic components of pulmonary vascular remodeling. Macrophages, T and B lymphocytes, and dendritic cells are present in the vascular lesions of PH, whether in idiopathic pulmonary arterial hypertension (PAH) or PAH related to more classical forms of inflammatory syndromes such as connective tissue diseases, human immunodeficiency virus (HIV), or other viral etiologies. Similarly, the presence of circulating chemokines and cytokines, viral protein components (e.g., HIV-1 Nef), and increased expression of growth (such as vascular endothelial growth factor and platelet-derived growth factor) and transcriptional (e.g., nuclear factor of activated T cells or NFAT) factors in these patients are thought to contribute directly to further recruitment of inflammatory cells and proliferation of smooth muscle and endothelial cells. Other processes, such as mitochondrial and ion channel dysregulation, seem to convey a state of cellular resistance to apoptosis; this has recently emerged as a necessary event in the pathogenesis of pulmonary vascular remodeling. Thus, the recognition of complex inflammatory disturbances in the vascular remodeling process offers potential specific targets for therapy and has recently led to clinical trials investigating, for example, the use of tyrosine kinase inhibitors. This paper provides an overview of specific inflammatory pathways involving cells, chemokines and cytokines, cellular dysfunctions, growth factors, and viral proteins, highlighting their potential role in pulmonary vascular remodeling and the possibility of future targeted therapy.

Defective pulmonary vascular remodeling in Smad8 mutant mice.

Pulmonary artery hypertension (PAH), a progressive, lethal condition that results in pathologic changes in the pulmonary arterial tree, eventually leads to right heart failure. Work identifying mutations in the Type II Bone morphogenetic protein (Bmp) receptor, BmpRII, in families with PAH has implicated Bmp-signaling in the pathogenesis of PAH. However, the effectors downstream of BmpRII in PAH remain unclear since BmpRII signals via Smad-dependent and independent mechanisms. We investigated Smad8 function, a divergent receptor regulated Smad downstream of Bmp-signaling, using gene targeting in mice. We show that Smad8 loss of function in adults resulted in characteristic changes in distal pulmonary arteries including medial thickening and smooth muscle hyperplasia that is observed in patients with PAH. Smad8 mutant pulmonary vasculature had upregulated Activin/Tgfbeta signaling and pathologic remodeling with aberrant Prx1 and Tenascin-C expression. A subset of Smad8 mutants had pulmonary adenomas uncovering a function for Smad8 in normal growth control. These findings implicate Smad8 in both pulmonary hypertension and lung tumorigenesis and support Smad8 as a candidate gene for PAH in humans.

ROCK controls matrix synthesis in vascular smooth muscle cells: coupling vasoconstriction to vascular remodeling.

Tenascin-C (TN-C) is an extracellular matrix (ECM) protein expressed within remodeling systemic and pulmonary arteries (PAs), where it supports vascular smooth muscle cell (SMC) proliferation. Previously, we showed that A10 SMCs cultivated on native type I collagen possess a spindle-shaped morphology and do not express TN-C, whereas those on denatured collagen possess a well-defined F-actin stress fiber network, a spread morphology, and they do express TN-C. To determine whether changes in cytoskeletal architecture control TN-C, SMCs on denatured collagen were treated with cytochalasin D, which decreased SMC spreading and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling effectors required for TN-C transcription. Next, to determine whether cell shape, dictated by the F-actin cytoskeleton, regulates TN-C, different geometries of SMCs (ranging from spread to round) were engineered on denatured collagen: as SMCs progressively rounded, ERK1/2 activity and TN-C transcription declined. Because RhoA and Rho kinase (ROCK) regulate cell morphology by controlling cytoskeletal architecture, we reasoned that these factors might also regulate TN-C. Indeed, SMCs on denatured collagen possessed higher levels of RhoA activity than those on native collagen, and blocking RhoA or ROCK activities attenuated SMC spreading, ERK1/2 activity, and TN-C expression in SMCs on denatured collagen. Thus, ROCK controls the configuration of the F-actin cytoskeleton and SMC shape in a manner that is permissive for ERK1/2-dependent production of TN-C. Finally, we showed that inhibition of ROCK activity suppresses SMC TN-C expression and disease progression in hypertensive rat PAs. Thus, in addition to its role in regulating vasoconstriction, ROCK also controls matrix production.

Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial hypertension.

Familial forms of human pulmonary arterial hypertension (FPAH) have been linked to mutations in bone morphogenetic protein (BMP) type II receptors (BMPR2s), yet the downstream targets of these receptors remain obscure. Here we show that pulmonary vascular lesions from patients harboring BMPR2 mutations express high levels of tenascin-C (TN-C), an extracellular matrix glycoprotein that promotes pulmonary artery (PA) smooth muscle cell (SMC) proliferation. To begin to define how TN-C is regulated, PA SMCs were cultured from normal subjects and from those with FPAH due to BMPR2 mutations. FPAH SMCs expressed higher levels of TN-C than normal SMCs. Similarly, expression of Prx1, a factor that drives TN-C transcription, was elevated in FPAH vascular lesions and SMCs derived thereof. Furthermore, Prx1 and TN-C promoter activities were significantly higher in FPAH vs. normal SMCs. To delineate how BMPR2s control TN-C, we focused on receptor (R)-Smads, downstream effectors activated by wild-type BMPR2s. Nuclear localization and phosphorylation of R-Smads was greater in normal vs. FPAH SMCs. As well, indirect blockade of R-Smad signaling with a kinase-deficient BMP receptor Ib upregulated TN-C in normal SMCs. Because ERK1/2 MAPKs inhibit the transcriptional activity of R-Smads, and because ERK1/2 promotes TN-C transcription, we determined whether ERK1/2 inhibits R-Smad signaling in FPAH SMCs and whether this activity is required for TN-C transcription. Indeed, ERK1/2 activity was greater in FPAH SMCs, and inhibition of ERK1/2 resulted in nuclear localization of R-Smads and inhibition of TN-C. These studies define a novel signaling network relevant to PAH underscored by BMPR2 mutations.

Fetal oxygen tension promotes tenascin-C-dependent lung branching morphogenesis.

Tenascin-C (TN-C) is a mesenchyme-derived extracellular matrix (ECM) glycoprotein required for fetal lung branching morphogenesis. Given that the low oxygen (O(2)) environment of the fetus is also essential for normal lung branching morphogenesis, we determined whether fetal O(2) tension supports this process by promoting TN-C expression. Initial studies showed that 15-day fetal rat lung explants cultured for 2 days at 3% O(2) not only branched well, but they also expressed higher levels of TN-C when compared to lungs maintained at 21% O(2), which branched poorly. Antisense oligonucleotide studies demonstrated that TN-C produced in response to 3% O(2) was essential for lung branching morphogenesis. As well, exogenous TN-C protein was shown to promote branching of lung epithelial rudiments cultured at 21% O(2). Because ECM-degrading proteinases are capable of catabolizing TN-C protein, we reasoned that 3% O(2) might promote TN-C deposition by limiting the activity of these enzymes within the fetal lung. Consistent with this idea, gelatin zymography showed that the activity of a 72-kDa gelatinase, identified as matrix metalloproteinase-2 (MMP-2), was lower at 3% O(2) vs. 21% O(2). Furthermore, pharmacologic inhibition of MMP-2 activity in fetal lung explants cultured at 21% O(2) resulted in increased TN-C deposition within the mesenchyme, as well as enhanced branching morphogenesis. Collectively, these studies indicate that fetal O(2) tension promotes TN-C-dependent lung epithelial branching morphogenesis by limiting the proteolytic turnover of this ECM component within the adjacent mesenchyme.

Development of occlusive neointimal lesions in distal pulmonary arteries of endothelin B receptor-deficient rats: a new model of severe pulmonary arterial hypertension.

BACKGROUND: Human pulmonary arterial hypertension (PAH) is characterized by proliferation of vascular smooth muscle and, in its more severe form, by the development of occlusive neointimal lesions. However, few animal models of pulmonary neointimal proliferation exist, thereby limiting a complete understanding of the pathobiology of PAH. Recent studies of the endothelin (ET) system demonstrate that deficiency of the ET(B) receptor predisposes adult rats to acute and chronic hypoxic PAH, yet these animals fail to develop neointimal lesions. Herein, we determined and thereafter showed that exposure of ET(B) receptor-deficient rats to the endothelial toxin monocrotaline (MCT) leads to the development of neointimal lesions that share hallmarks of human PAH. METHODS AND RESULTS: The pulmonary hemodynamic and morphometric effects of 60 mg/kg MCT in control (MCT(+/+)) and ET(B) receptor-deficient (MCT(sl/sl)) rats at 6 weeks of age were assessed. MCT(sl/sl) rats developed more severe PAH, characterized by elevated pulmonary artery pressure, diminished cardiac output, and right ventricular hypertrophy. In MCT(sl/sl) rats, morphometric evaluation revealed the presence of neointimal lesions within small distal pulmonary arteries, increased medial wall thickness, and decreased arterial-to-alveolar ratio. In keeping with this, barium angiography revealed diminished distal pulmonary vasculature of MCT(sl/sl) rat lungs. Cells within neointimal lesions expressed smooth muscle and endothelial cell markers. Moreover, cells within neointimal lesions exhibited increased levels of proliferation and were located in a tissue microenvironment enriched with vascular endothelial growth factor, tenascin-C, and activated matrix metalloproteinase-9, factors already implicated in human PAH. Finally, assessment of steady state mRNA showed that whereas expression of ET(B) receptors was decreased in MCT(sl/sl) rat lungs, ET(A) receptor expression increased. CONCLUSIONS: Deficiency of the ET(B) receptor markedly accelerates the progression of PAH in rats treated with MCT and enhances the appearance of cellular and molecular markers associated with the pathobiology of PAH. Collectively, these results suggest an overall antiproliferative effect of the ET(B) receptor in pulmonary vascular homeostasis.

Paired-related homeobox gene Prx1 is required for pulmonary vascular development.

Herein, we show that the paired-related homeobox gene, Prx1, is required for lung vascularization. Initial studies revealed that Prx1 localizes to differentiating endothelial cells (ECs) within the fetal lung mesenchyme, and later within ECs forming vascular networks. To begin to determine whether Prx1 promotes EC differentiation, fetal lung mesodermal cells were transfected with full-length Prx1 cDNA, resulting in their morphological transformation to an endothelial-like phenotype. In addition, Prx1-transformed cells acquired the ability to form vascular networks on Matrigel. Thus, Prx1 might function by promoting pulmonary EC differentiation within the fetal lung mesoderm, as well as their subsequent incorporation into vascular networks. To understand how Prx1 participates in network formation, we focused on tenascin-C (TN-C), an extracellular matrix (ECM) protein induced by Prx1. Immunocytochemistry/histochemistry showed that a TN-C-rich ECM surrounds Prx1-positive pulmonary vascular networks both in vivo and in tissue culture. Furthermore, antibody-blocking studies showed that TN-C is required for Prx1-dependent vascular network formation on Matrigel. Finally, to determine whether these results were relevant in vivo, we examined newborn Prx1-wild-type (+/+) and Prx1-null (-/-) mice and showed that Prx1 is critical for expression of TN-C and lung vascularization. These studies provide a framework to understand how Prx1 controls EC differentiation and their subsequent incorporation into functional pulmonary vascular networks.

Homeobox genes in pulmonary vascular development and disease.

Determining how the pulmonary vascular system is formed, maintained, or disrupted during development and disease represents a major challenge in contemporary lung biology. Whereas it is appreciated that cellular proliferation, differentiation, migration, and apoptosis need to be carefully controlled in order to attain pulmonary vascular homeostasis, knowledge of the underlying cellular and molecular mechanisms involved remains surprisingly limited. Because homeobox genes represent master regulators of organogenesis and tissue patterning, it is likely that these transcription factors play a critical role in the formation of blood vessels within the lung, as well as in pathologic states in which the highly ordered structure of the pulmonary vascular tree is compromised. The aim of this review is to discuss some of the known functions of homeobox genes in the vasculature, and to extrapolate these findings to their potential roles in developing and diseased pulmonary vessels.