YF17D-based vaccines - standing on the shoulders of a giant.

Live-attenuated yellow fever vaccine (YF17D) was developed in the 1930s as the first ever empirically derived human vaccine. Ninety years later, it is still a benchmark for vaccines made today. YF17D triggers a particularly broad and polyfunctional response engaging multiple arms of innate, humoral and cellular immunity. This unique immunogenicity translates into an extraordinary vaccine efficacy and outstanding longevity of protection, possibly by single-dose immunization. More recently, progress in molecular virology and synthetic biology allowed engineering of YF17D as a powerful vector and promising platform for the development of novel recombinant live vaccines, including two licensed vaccines against Japanese encephalitis and dengue, even in paediatric use. Likewise, numerous chimeric and transgenic preclinical candidates have been described. These include prophylactic vaccines against emerging viral infections (e.g. Lassa, Zika and SARS-CoV-2) and parasitic diseases (e.g. malaria), as well as therapeutic applications targeting persistent infections (e.g. HIV and chronic hepatitis), and cancer. Efforts to overcome historical safety concerns and manufacturing challenges are ongoing and pave the way for wider use of YF17D-based vaccines. In this review, we summarize recent insights regarding YF17D as vaccine platform, and how YF17D-based vaccines may complement as well as differentiate from other emerging modalities in response to unmet medical needs and for pandemic preparedness.

Distinct and dynamic activation profiles of circulating dendritic cells and monocytes in mild COVID-19 and after yellow fever vaccination.

Dysregulation of the myeloid cell compartment is a feature of severe disease in hospitalized COVID-19 patients. Here, we investigated the response of circulating dendritic cell (DC) and monocyte subpopulations in SARS-CoV-2 infected outpatients with mild disease and compared it to the response of healthy individuals to yellow fever vaccine virus YF17D as a model of a well-coordinated response to viral infection. In SARS-CoV-2-infected outpatients circulating DCs were persistently reduced for several weeks whereas after YF17D vaccination DC numbers were decreased temporarily and rapidly replenished by increased proliferation until 14 days after vaccination. The majority of COVID-19 outpatients showed high expression of CD86 and PD-L1 in monocytes and DCs early on, resembling the dynamic after YF17D vaccination. In a subgroup of patients, low CD86 and high PD-L1 expression were detected in monocytes and DCs coinciding with symptoms, higher age, and lower lymphocyte counts. This phenotype was similar to that observed in severely ill COVID-19 patients, but less pronounced. Thus, prolonged reduction and dysregulated activation of blood DCs and monocytes were seen in a subgroup of symptomatic non-hospitalized COVID-19 patients while a transient coordinated activation was characteristic for the majority of patients with mild COVID-19 and the response to YF17D vaccination.

Interleukin-17D produced by alveolar epithelial type II cells alleviates LPS-induced acute lung injury via the Nrf2 pathway.

BACKGROUND: Sepsis engenders an imbalance in the body's inflammatory response, with cytokines assuming a pivotal role in its progression. A relatively recent addition to the interleukin-17 family, denominated interleukin-17D (IL-17D), is notably abundant within pulmonary confines. Nevertheless, its implication in sepsis remains somewhat enigmatic. The present study endeavors to scrutinize the participation of IL-17D in sepsis-induced acute lung injury (ALI). METHODS: The levels of IL-17D in the serum and bronchoalveolar lavage fluid (BALF) of both healthy cohorts and septic patients were ascertained through an ELISA protocol. For the creation of a sepsis-induced ALI model, intraperitoneal lipopolysaccharide (LPS) injections were administered to male C57/BL6 mice. Subsequently, we examined the fluctuations and repercussions associated with IL-17D in sepsis-induced ALI, probing its interrelation with nuclear factor erythroid 2-related factor 2 (Nrf2), alveolar epithelial permeability, and heme oxygenase-1. RESULTS: IL-17D levels exhibited significant reduction both in the serum and BALF of septic patients (P<0.001). Similar observations manifested in mice subjected to LPS-induced acute lung injury (ALI) (P=0.002). Intraperitoneal administration of recombinant interleukin 17D protein (rIL-17D) prompted increased expression of claudin 18 and concomitant enhancement of alveolar epithelial permeability, thus, culminating in improved lung injury (P<0.001). Alveolar epithelial type II (ATII) cells were identified as the source of IL-17D, regulated by Nrf2. Furthermore, a deficiency in HO-1 yielded elevated IL-17D levels (P=0.004), albeit administration of rIL-17D ameliorated the exacerbated pulmonary damage resulting from HO-1 deficiency. CONCLUSION: Nrf2 fosters IL-17D production within AT II cells, thereby conferring a protective role in sepsis-induced ALI.

Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells.

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-ß (DEFB1)] and some inflammatory genes such as IL-1ß, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost.

Persistence of Neutralizing Antibody Responses Among Yellow Fever Virus 17D Vaccinees Living in a Nonendemic Setting.

BACKGROUND: The once-in-a-lifetime recommendation for vaccination against yellow fever virus (YFV) has been controversial, leading to increased scrutiny of the durability of immunity after 17D vaccination. METHODS: This is a cross-sectional analysis of 17D vaccinees living in nonendemic Portland, Oregon. Neutralization assays were used to determine YFV immunity. The relationships between 17D immunity and vaccination history, demographics, and travel were evaluated using nominal logistic regression. RESULTS: Seventy-one of 92 (77.2%) subjects were YFV seropositive (90 percent plaque reduction neutralization test ≥1:10) at all timepoints, and 24 of 38 (63.8%) were YFV seropositive at ≥10 years after single-dose vaccination. No relationship was found between YFV immunity and time in endemic countries, other flavivirus immunity, or demographics. Subjects were most likely to become seronegative between 3 and 12 years postvaccination (logistic regression, odds ratio [OR] = 1.75; 95% confidence interval [CI], 1.12-2.73). A comparison of our results and 4 previous studies of YFV nonendemic vaccinees found that overall, 79% (95% CI, 70%-86%) of vaccinees are likely to be seropositive ≥10 years postvaccination. CONCLUSIONS: These results suggest that 1 in 5 17D vaccinees will lack neutralizing antibodies at ~10 years postvaccination, and a booster vaccination should be considered for nonendemic vaccinees before travel to regions where there is a high risk of YFV transmission.

L-selectin activation regulates Rho GTPase activity via Ca+2 influx in Sertoli cell line, ASC-17D cells.

In seminiferous epithelium, tight junctions (TJs) between adjacent Sertoli cells constitute the blood-testis barrier and must change synchronically for germ cells to translocate from the basal to the adluminal compartment during the spermatogenic cycle. Rho GTPase activation through stimulation with specific L-selectin ligands has been proposed to modulate tight junctional dynamics. However, little is known regarding the role of Ca+2 dynamics in Sertoli cell and how Ca+2 relays L-selectin signals to modulate Rho GTPase activity in Sertoli cells, thus prompting us to investigate the Ca+2 flux induced by L-selectin ligand in ASC-17D cells. Using fluorescent real-time image, we first demonstrated the increase of intracellular Ca+2 level following L-selectin ligand stimulation. This Ca+2 increase was inhibited in ASC-17D cells pretreated with nifedipine, the L-type voltage-operated Ca+2 channel (VOCC) blocker, but not mibefradil, the T-type VOCC blocker. We then demonstrated the up-regulation of Rho and Rac1 in ASC-17D cells following the administration of L-selectin ligand, and the pre-treatment with nifedipine, but not mibefradil, prior to L-selectin ligand-binding abolished the activation of both Rho and Rac1. Together, we conclude that the activation of L-selectin induces Ca+2 influx through the L-type VOCC, which up-regulates Rho and Rac1 proteins, in ASC-17D cells.

IL-17D: A Less Studied Cytokine of IL-17 Family.

The interleukin-17 (IL-17) family is a relatively new family of cytokines consisting of 6 related factors (IL-17A-IL-17F), while the receptor family consists of 5 members: IL-17RA-IL-17RE. IL-17A is the prototype member of this family, which is also the signature cytokine of T helper 17 (Th17) cells. Th17 cells are involved in the development of autoimmune disease, inflammation, and tumors. Although IL-17D is similar to IL-17A in its ability to induce inflammatory cytokine production, there are fewer studies on IL-17D. Recently, the role of IL-17D in tumors and infections has attracted our attention. Some knowledge of function of IL-17D has been gained by studies using nonmammalian species. In this review, we introduce the structural characteristics, expression patterns, and biological characteristics of IL-17D along with its potential function in the pathogenesis of disease.

VIO: ontology classification and study of vaccine responses given various experimental and analytical conditions.

BACKGROUND: Different human responses to the same vaccine were frequently observed. For example, independent studies identified overlapping but different transcriptomic gene expression profiles in Yellow Fever vaccine 17D (YF-17D) immunized human subjects. Different experimental and analysis conditions were likely contributed to the observed differences. To investigate this issue, we developed a Vaccine Investigation Ontology (VIO), and applied VIO to classify the different variables and relations among these variables systematically. We then evaluated whether the ontological VIO modeling and VIO-based statistical analysis would contribute to the enhanced vaccine investigation studies and a better understanding of vaccine response mechanisms. RESULTS: Our VIO modeling identified many variables related to data processing and analysis such as normalization method, cut-off criteria, software settings including software version. The datasets from two previous studies on human responses to YF-17D vaccine, reported by Gaucher et al. (2008) and Querec et al. (2009), were re-analyzed. We first applied the same LIMMA statistical method to re-analyze the Gaucher data set and identified a big difference in terms of significantly differentiated gene lists compared to the original study. The different results were likely due to the LIMMA version and software package differences. Our second study re-analyzed both Gaucher and Querec data sets but with the same data processing and analysis pipeline. Significant differences in differential gene lists were also identified. In both studies, we found that Gene Ontology (GO) enrichment results had more overlapping than the gene lists and enriched pathway lists. The visualization of the identified GO hierarchical structures among the enriched GO terms and their associated ancestor terms using GOfox allowed us to find more associations among enriched but often different GO terms, demonstrating the usage of GO hierarchical relations enhance data analysis. CONCLUSIONS: The ontology-based analysis framework supports standardized representation, integration, and analysis of heterogeneous data of host responses to vaccines. Our study also showed that differences in specific variables might explain different results drawn from similar studies.

Development of a Real-Time Reverse Transcription-PCR Assay for Global Differentiation of Yellow Fever Virus Vaccine-Related Adverse Events from Natural Infections.

Yellow fever (YF) is a reemerging public health threat, with frequent outbreaks prompting large vaccination campaigns in regions of endemicity in Africa and South America. Specific detection of vaccine-related adverse events is resource-intensive, time-consuming, and difficult to achieve during an outbreak. To address this, we have developed a highly transferable rapid yellow fever virus (YFV) vaccine-specific real-time reverse transcription-PCR (RT-PCR) assay that distinguishes vaccine from wild-type lineages. The assay utilizes a specific hydrolysis probe that includes locked nucleic acids to enhance specific discrimination of the YFV17D vaccine strain genome. Promisingly, sensitivity and specificity analyses reveal this assay to be highly specific to vaccine strain(s) when tested on clinical samples and YFV cell culture isolates of global origin. Taken together, our data suggest the utility of this assay for use in laboratories of varied capacity for the identification and differentiation of vaccine-related adverse events from wild-type infections of both African and South American origin.

The ancient cytokine IL-17D is regulated by Nrf2 and mediates tumor and virus surveillance.

Early stage immune responses can dictate the severity and outcome of inflammatory processes such as tumor growth and viral infection. Cytokines such as the interleukin 17 (IL-17) family and cellular stress defense (e.g., anti-oxidant) pathways have evolved early and regulate disease surveillance in vertebrates and invertebrates as far back as Caenorhabditis elegans. Our group has recently found a new role for nuclear factor erythroid-derived 2-like 2 (Nrf2) in regulating early anti-cancer immune responses by inducing IL-17D and recruiting natural killer (NK) cells. In this Cytokine Stimulus, we discuss recent findings that encourage boosting the Nrf2/IL-17D/NK cell axis for the treatment of cancer and viral infection.

Peritoneal bacterial infection repressed the expression of IL17D in Siberia sturgeon a chondrostean fish in the early immune response.

IL17s are pro-inflammatory cytokines that play important roles in host fighting against extracellular bacteria and auto-immune and allergic diseases. IL17D is believed to be the most ancient IL17 member and its functions are far from clarity. Although it has been found in invertebrates, jawless fish, teleosts, and tetrapods, it has not been described in chondrostean fish. Moreover, there are discrepancies concerning its expression pattern in these animals. In this study, we cloned and characterized the cDNA of il17d in Siberia sturgeon (Acipenser baerii), a chondrostean fish and commercially important species in aquaculture. The sturgeon il17d cDNA encodes a deduced protein of 210aa. The classical characteristics of IL17, such as IL17 domain, cysteine and serine residues importantly for cystine-knot formation, and signal peptide, were observed in sturgeon IL17D. Phylogenetic analysis and multiple alignment suggest it is a counterpart of mammalian IL17D. However, in vivo studies demonstrated that the expression pattern of sturgeon il17d mRNA is different from that of other teleosts and jawless fish, and in most cases its expression was down-regulated at the early time points and gradually increasing at late time points when sturgeon were challenged with bacteria (Aernomas hydrophila or Staphylococcus aureus). The In vitro study by using primary spleen cells stimulated with polyI:C revealed a similar expression pattern to that in vivo studies, while the stimulation with ß-glucan or LPS, which normally induced expression of il17d mRNA in target cells in vitro in other animals, did not show apparent changes in the expression of il17d mRNA. The results of present study indicated sturgeon IL17D may possess some different characteristics from its counterparts of other fish and invertebrates in the immune response, and may contribute to the understanding of IL17D functions in evolution as well as the potential use in sturgeon aquaculture.

Comparison of the live attenuated yellow fever vaccine 17D-204 strain to its virulent parental strain Asibi by deep sequencing.

BACKGROUND: The first comparison of a live RNA viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (YFV) live vaccine strain 17D-204 and its wild-type parental strain, Asibi. METHODS: The YFV 17D-204 vaccine genome was compared to that of the parental strain Asibi by massively parallel methods. Variability was compared on multiple scales of the viral genomes. A modeled exploration of small-frequency variants was performed to reconstruct plausible regions of mutational plasticity. RESULTS: Overt quasispecies diversity is a feature of the parental strain, whereas the live vaccine strain lacks diversity according to multiple independent measurements. A lack of attenuating mutations in the Asibi population relative to that of 17D-204 was observed, demonstrating that the vaccine strain was derived by discrete mutation of Asibi and not by selection of genomes in the wild-type population. CONCLUSIONS: Relative quasispecies structure is a plausible correlate of attenuation for live viral vaccines. Analyses such as these of attenuated viruses improve our understanding of the molecular basis of vaccine attenuation and provide critical information on the stability of live vaccines and the risk of reversion to virulence.

Electron transport chain capacity expands yellow fever vaccine immunogenicity.

Vaccination has successfully controlled several infectious diseases although better vaccines remain desirable. Host response to vaccination studies have identified correlates of vaccine immunogenicity that could be useful to guide development and selection of future vaccines. However, it remains unclear whether these findings represent mere statistical correlations or reflect functional associations with vaccine immunogenicity. Functional associations, rather than statistical correlates, would offer mechanistic insights into vaccine-induced adaptive immunity. Through a human experimental study to test the immunomodulatory properties of metformin, an anti-diabetic drug, we chanced upon a functional determinant of neutralizing antibodies. Although vaccine viremia is a known correlate of antibody response, we found that in healthy volunteers with no detectable or low yellow fever 17D viremia, metformin-treated volunteers elicited higher neutralizing antibody titers than placebo-treated volunteers. Transcriptional and metabolomic analyses collectively showed that a brief course of metformin, started 3 days prior to YF17D vaccination and stopped at 3 days after vaccination, expanded oxidative phosphorylation and protein translation capacities. These increased capacities directly correlated with YF17D neutralizing antibody titers, with reduced reactive oxygen species response compared to placebo-treated volunteers. Our findings thus demonstrate a functional association between cellular respiration and vaccine-induced humoral immunity and suggest potential approaches to enhancing vaccine immunogenicity.

Post-Lie algebra structures for perfect Lie algebras.

We study the existence of post-Lie algebra structures on pairs of Lie algebras ( g , n ) , where one of the algebras is perfect non-semisimple, and the other one is abelian, nilpotent non-abelian, solvable non-nilpotent, simple, semisimple non-simple, reductive non-semisimple or complete non-perfect. We prove several nonexistence results, but also provide examples in some cases for the existence of a post-Lie algebra structure. Among other results we show that there is no post-Lie algebra structure on ( g , n ) , where g is perfect non-semisimple, and n is s l 3 ( C ) . We also show that there is no post-Lie algebra structure on ( g , n ) , where g is perfect and n is reductive with a 1-dimensional center.

Parallel Multifactorial Process Optimization and Intensification for High-Yield Production of Live YF17D-Vectored Zika Vaccine.

The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEKDyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.

Yellow fever vaccine: comparison of the neurovirulence of new 17D-204 Stamaril™ seed lots and RK 168-73 strain.

The neurovirulence of two new candidate 17D-204 Stamaril™ working seed lots and that of two reference preparations were compared. The Stamaril™ working seed lots have been used for more than twenty years for the manufacturing of vaccines of acceptable safety and efficacy. The preparation designated RK 168-73 and provided by the Robert Koch Institute was used as a reference. It was confirmed that RK 168-73 strain was not a good virus control in our study because it has a very low neurovirulence regarding both the clinical and histopathological scores in comparison with Stamaril™ strain and is not representative of a vaccine known to be satisfactory in use. The results were reinforced by the phenotypic characterization by plaque assay demonstrating that RK 168-73 was very different from the Stamaril™ vaccine, and by sequencing results showing 4 mutations between Stamaril™ and RK 168-73 viruses leading to amino acid differences in the NS4B and envelop proteins.

Characterization of Live-Attenuated Powassan Virus Vaccine Candidates Identifies an Efficacious Prime-Boost Strategy for Mitigating Powassan Virus Disease in a Murine Model.

Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease.

ROS-activated MAPK/ERK pathway regulates crosstalk between Nrf2 and Hif-1α to promote IL-17D expression protecting the intestinal epithelial barrier under hyperoxia.

Reactive oxygen species (ROS) damage to the intestinal barrier is a side effect of prolonged hyperoxia therapy in neonates, which impairs growth and development of the intestine and promotes intestinal diseases. However, the research on clinical prevention and treatment is lacking. Therefore, we investigated the molecular mechanisms of the neonate intestinal response against hyperoxia-derived ROS to find targets for intestinal barrier damage prevention. Human intestinal epithelial cells were incubated under hyperoxia (85% oxygen) to build an in vitro model. ROS and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway were inhibited to detect the MAPK/ERK pathway, nuclear factor erythroid factor 2-related factor 2 (Nrf2), hypoxia-inducible factor-1α (Hif-1α), and interleukin-17D (IL-17D) expression. Nrf2 was inhibited to detect Hif-1α and IL-17D expression. Hif-1α was inhibited to detect Nrf2, IL-17D, and tight junction proteins expression and apoptosis. Cells were treated with human recombinant IL-17D to detect TNF-α, IL-1ß, IL-10, and tight junction proteins expression. ROS, Nrf2, Hif-1α, and IL-17D were upregulated and the MAPK/ERK pathway was activated under hyperoxia. But ROS inhibition downregulated the MAPK/ERK pathway, Nrf2, Hif-1α, and IL-17D. MAPK/ERK pathway inhibition downregulated Nrf2, Hif-1α, and IL-17D. Nrf2 inhibition downregulated Hif-1α and IL-17D. Hif-1α inhibition downregulated Nrf2, IL-17D, tight junction proteins, and exacerbated apoptosis. The recombinant IL-17D downregulated TNF-α, IL-1ß, but upregulated IL-10 and tight junction proteins. We concluded that Hyperoxia-generated ROS activated the MAPK/ERK pathway to regulate Nrf2, Hif-1α, and IL-17D expression. Nrf2 and Hif-1α were interdependent and promoted IL-17D. Importantly, Hif-1α and IL-17D expression protected the intestinal epithelial barrier.

Brain-wide circuit-specific targeting of astrocytes.

Astrocytes are integral components of brain circuitry. They enwrap synapses, react to neuronal activity, and regulate synaptic transmission. Astrocytes are heterogeneous and exhibit distinct features and functions in different circuits. Selectively targeting the astrocytes associated with a given neuronal circuit would enable elucidation of their circuit-specific functions but has been technically challenging to date. Recently, we constructed anterograde transneuronal viral vectors based on yellow fever vaccine YFV-17D. Among them, the replication-incompetent YFVΔNS1-Cre can selectively turn on reporter genes in postsynaptic neurons if the viral gene NS1 is expressed in postsynaptic neurons. Here we show that without exogenous expression of NS1 at the postsynaptic sites, locally injected YFVΔNS1-Cre selectively turns on reporter genes in astrocytes in downstream brain regions. The targeting of astrocytes can occur across the whole brain but is specific for the neuronal circuits traced. Therefore, YFVΔNS1-Cre provides a tool for selective genetic targeting of astrocytes to reveal their circuit-specific roles.

Antibacterial Properties and Efficacy of LL-37 Fragment GF-17D3 and Scolopendin A2 Peptides Against Resistant Clinical Strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii In Vitro and In Vivo Model Studies.

Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii have emerged as major clinical threats owing to the increasing prevalence of ventilator-associated pneumonia caused by multidrug-resistant or extensively drug-resistant strains. The present study aimed to assess the antibacterial effects and efficacy of LL-37 fragment GF-17D3 and synthetic Scolopendin A2 peptides against resistant clinical strains in vitro and in vivo models. P. aeruginosa, S. aureus, and A. baumannii were isolated from clinical infections. Their antibiotic resistance and minimum inhibitory concentration were assessed. LL-37 fragment GF-17D3 peptide was selected from available databases. Scolopendin A2 peptide's 6th amino acid (proline) was substituted with lysine and peptides and MICs were determined. The biofilm inhibitory activity was quantified at sub MIC concentrations. Synergetic effects of Scolopendin A2 and imipenem were assessed by checkerboard. After mice nasal infection with P. aeruginosa, peptides LD50 was determined. Isolates harbored complete resistance toward the majority of antibiotics and MIC values ranged between 1 and > 512 µg/ml. The majority of isolates exhibited strong biofilm activity. Synthetic peptides showed lower MIC values than antibiotic agents and the lowest MIC values were obtained for synthetic peptides in combination with antibiotics. The Synergisms effect of Scolopendin A2 with imipenem was also determined. Scolopendin A2 was found to have antibacterial efficacy against P. aeruginosa, S. aureus, and A. baumannii with MIC 64 µg/ml, 8 µg/ml, and 16 µg/ml, respectively, and LL37 showed antibacterial efficacy against P. aeruginosa, S. aureus, and A. baumannii with MIC 128 µg/ml, 32 µg/ml, and 32 µg/ml, respectively. Both AMPs decreased biofilms by ≥ 96% at 1 × MIC. The biofilm inhibitory activity was measured at sub MIC concentrations of the peptides and the results demonstrated that Scolopendin A2 exhibited anti-biofilm activity at 1/4 × MIC and 1/2 × MIC concentrations was 47.9 to 63.8%, although LL37 among 1/4 × MIC and 1/2 × MIC concentrations was 21.3 to 49.6% against three pathogens. The combination of Scolopendin A2 and antibiotics demonstrated synergistic activity-resistant strains with FIC values ≤ 0.5 for three pathogens, while LL37 and antibiotics showed synergistic activity FIC values ≤ 0.5 for only P. aeruginosa. Infection model Scolopendin A2 with Imipenem (2 × MIC) was efficacious in vivo, with a 100% survival rate following treatment at 2 × MIC after 120 h. The mRNA expression of biofilm-related genes was decreased for both peptides. Synthesis Scolopendin A2 decreased the expression of biofilm formation genes compared to the control group. Synthetic Scolopendin A2 exhibits antimicrobial activity without causing toxicity on the human epithelial cell line. Based on our findings, it seems that synthetic Scolopendin A2 is an appropriate antimicrobial source. That could be a promising option in combination with antibiotics for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant bacteria. Nevertheless, additional experiments are required to assess another potential of this novel AMP.