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The emergence of rapidly spreading variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a major challenge to vaccines' protective efficacy. Intramuscular (IM) vaccine administration induces short-lived immunity but does not prevent infection and transmission. New vaccination strategies are needed to extend the longevity of vaccine protection, induce mucosal and systemic immunity and prevent viral transmission. The intranasal (IN) administration of the VSV-ΔG-spike vaccine candidate directly to mucosal surfaces yielded superior mucosal and systemic immunity at lower vaccine doses. Compared to IM vaccination in the K18-hACE2 model, IN vaccination preferentially induced mucosal IgA and T-cells, reduced the viral load at the site of infection, and ameliorated disease-associated brain gene expression. IN vaccination was protective even one year after administration. As most of the world population has been vaccinated by IM injection, we demonstrate the potential of a heterologous IM + IN vaccination regimen to induce mucosal immunity while maintaining systemic immunity. Furthermore, the IM + IN regimen prevented virus transmission in a golden Syrian hamster co-caging model. Taken together, we show that IN vaccination with VSV-ΔG-spike, either as a homologous IN + IN regimen or as a boost following IM vaccination, has a favorable potential over IM vaccination in inducing efficient mucosal immunity, long-term protection and preventing virus transmission.
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OBJECTIVE: Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. DATA DESCRIPTION: We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.
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Burkholderia pseudomallei , Genoma Bacteriano , Melioidose , Filogenia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/patogenicidade , Melioidose/microbiologia , Melioidose/epidemiologia , Tailândia/epidemiologia , Humanos , Genoma Bacteriano/genética , Índia , Animais , Israel/epidemiologia , Virulência/genética , Camundongos , Sequenciamento Completo do GenomaRESUMO
The diagnosis of Q fever is challenging due to nonspecific symptoms and negative standard blood culture results. Serological testing through immunofluorescence assay (IFA) is the most commonly used method for diagnosing this disease. Polymerase chain reaction (PCR) tests can also be used to detect bacterial DNA if taken at an appropriate time. Once the presence of bacteria is confirmed in a sample, an enrichment step is required before characterizing it through sequencing. Cultivating C. burnetii is challenging as it can only be isolated by inoculation into cell culture, embryonated eggs, or animals. In this article, we describe the isolation of C. burnetii from a valve specimen in Vero cells. We conducted genome sequencing and taxonomy profiling of this isolate and were able to determine its taxonomic affiliation. Furthermore, Multispacer sequence typing (MST) analysis suggests that the infection originated from a local strain of C. burnetii found around northern Israel and Lebanon. This novel strain belongs to a previously described genotype MST6, harboring the QpRS plasmid, never reported in Israel.
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The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms.
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Since the emergence of the original SARS-CoV-2, several variants were described, raising questions as to the ability of recently developed vaccine platforms to induce immunity and provide protection against these variants. Here, we utilized the K18-hACE2 mouse model to show that VSV-ΔG-spike vaccination provides protection against several SARS-CoV-2 variants: alpha, beta, gamma, and delta. We show an overall robust immune response, regardless of variant identity, leading to reduction in viral load in target organs, prevention of morbidity and mortality, as well as prevention of severe brain immune response, which follows infection with various variants. Additionally, we provide a comprehensive comparison of the brain transcriptomic profile in response to infection with different variants of SARS-CoV-2 and show how vaccination prevents these disease manifestations. Taken together, these results highlight the robust VSV-ΔG-spike protective response against diverse SARS-CoV-2 variants, as well as its promising potential against newly arising variants.
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Vacinas contra COVID-19 , COVID-19 , Animais , Humanos , Camundongos , COVID-19/prevenção & controle , SARS-CoV-2/genética , Glicoproteína da Espícula de CoronavírusRESUMO
Virus-induced CNS diseases impose a considerable human health burden worldwide. For many viral CNS infections, neither antiviral drugs nor vaccines are available. In this study, we examined whether the synthesis of glycosphingolipids, major membrane lipid constituents, could be used to establish an antiviral therapeutic target. We found that neuroinvasive Sindbis virus altered the sphingolipid levels early after infection in vitro and increased the levels of gangliosides GA1 and GM1 in the sera of infected mice. The alteration in the sphingolipid levels appears to play a role in neuroinvasive Sindbis virus replication, as treating infected cells with UDP-glucose ceramide glucosyltransferase (UGCG) inhibitors reduced the replication rate. Moreover, the UGCG inhibitor GZ-161 increased the survival rates of Sindbis-infected mice, most likely by reducing the detrimental immune response activated by sphingolipids in the brains of Sindbis virus-infected mice. These findings suggest a role for glycosphingolipids in the host immune response against neuroinvasive Sindbis virus and suggest that UGCG inhibitors should be further examined as antiviral therapeutics for viral infections of the CNS.
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OBJECTIVE: As part of a research aiming at presenting an alternative approach for rapid determination of antimicrobial susceptibility by quantification of changes in expression levels of specific marker genes and gene sets, cultures of the virulent bacterial strain Francisella tularensis SchuS4 were grown in the presence of inhibitory/sub-inhibitory concentrations of either ciprofloxacin or doxycycline and their transcriptomic profiles were elucidated using differential expression analysis followed by functional annotation. DATA DESCRIPTION: RNA sequencing was performed to identify differentially expressed genes (DEGs) in response to exposure of F. tularensis SchuS4 to either ciprofloxacin or doxycycline, the antibiotics of choice for Tularemia therapy. Accordingly, RNA samples were collected 2 h post antibiotic exposure and subjected to RNA sequence analysis. Transcriptomic quantification of RNA representing duplicated samples generated highly similar gene expression data. Exposure to sub-inhibitory concentration [0.5 x MIC (minimal inhibitory concentration)] of doxycycline or ciprofloxacin modulated the expression of 237 or 8 genes, respectively, while exposure to an inhibitory concentration (1 x MIC) resulted in the modulation of 583 or 234 genes, respectively. Amongst the genes modulated upon doxycycline exposure upregulation of 31 genes encoding for translation-functions could be distinguished, as well as downregulation of 14 genes encoding for functions involved in DNA transcription and repair. Ciprofloxacin exposure impacted differently the RNA sequence profile of the pathogen, resulting in upregulation of 27 genes encoding mainly DNA replication and repair functions, transmembrane transporters and molecular chaperons. In addition, 15 downregulated genes were involved in translation processes.
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Doxiciclina , Francisella tularensis , Doxiciclina/farmacologia , Francisella tularensis/genética , Ciprofloxacina/farmacologia , Transcriptoma/genética , Antibacterianos/farmacologia , RNARESUMO
Microbial adaptation to changing environmental conditions is frequently mediated by hypermutable sequences. Here we demonstrate that such a hypermutable hotspot within a gene encoding a flagellar unit of Paenibacillus glucanolyticus generated spontaneous non-swarming mutants with increased stress resistance. These mutants, which survived conditions that eliminated wild-type cultures, could be carried by their swarming siblings when the colony spread, consequently increasing their numbers at the spreading edge. Of interest, the hypermutable nature of the aforementioned sequence enabled the non-swarming mutants to serve as "seeds" for a new generation of wild-type cells through reversion of the mutation. Using a mathematical model, we examined the survival dynamics of P. glucanolyticus colonies under fluctuating environments. Our experimental and theoretical results suggest that the non-swarming, stress-resistant mutants can save the colony from extinction. Notably, we identified this hypermutable sequence in flagellar genes of additional Paenibacillus species, suggesting that this phenomenon could be wide-spread and ecologically important.
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Hyper-immune antisera from large mammals, in particular horses, are routinely used for life-saving anti-intoxication intervention. While highly efficient, the use of these immunotherapeutics is complicated by possible recipient reactogenicity and limited availability. Accordingly, there is an urgent need for alternative improved next-generation immunotherapies to respond to this issue of high public health priority. Here, we document the development of previously unavailable tools for equine antibody engineering. A novel primer set, EquPD v2020, based on equine V-gene data, was designed for efficient and accurate amplification of rearranged horse antibody V-segments. The primer set served for generation of immune phage display libraries, representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins. Highly specific scFv clones were selected and expressed as full-length antibodies, carrying equine V-genes and human Gamma1/Lambda constant genes, to be referred as "Centaur antibodies". Preliminary assessment in a murine model of botulism established their therapeutic potential. The experimental approach detailed in the current report, represents a valuable tool for isolation and engineering of therapeutic equine antibodies.
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Anticorpos , Região Variável de Imunoglobulina , Animais , Anticorpos/genética , Técnicas de Visualização da Superfície Celular , Cavalos , Humanos , Região Variável de Imunoglobulina/genética , Mamíferos , Camundongos , Neurotoxinas , Proteínas Recombinantes/genéticaRESUMO
As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population's immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs' clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5'-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.
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Mpox , Sequenciamento por Nanoporos , Orthopoxvirus , Varíola , Vírus da Varíola , Diagnóstico Diferencial , Humanos , Mpox/epidemiologia , Monkeypox virus/genética , Varíola/diagnóstico , Vírus da Varíola/genéticaRESUMO
OBJECTIVE: As part of a research aiming at the isolation of bacteria secreting growth inhibiting compounds, cultures of Francisella tularensis were implanted in environmental samples and monitored for inhibition zones on agar. Two antibiotic-like secreting bacteria were isolated, their genomic sequence was deciphered and taxonomic profiling analysis classified them as belonging to the Pantoea genus. DATA DESCRIPTION: Two bacterial isolates exhibiting growth inhibition zones to F. tularensis (LVS) were analyzed using the Oxford Nanopore Technology (ONT). Preliminary de novo assembly of the reads was performed, followed by taxonomic profiling based on Multi Locus Sequence Analysis (MLSA) and implementation of the Average Nucleotide Identity (ANI) measure. The genomic sequences resulted in the identification of two different Pantoea species, denoted EnvD and EnvH. Subsequent de novo genome assembly generated 5 and 10 contigs for EnvD and EnvH, respectively. The largest contig (4,008,183 bps and 3,740,753 bps for EnvD and EnvH, respectively), overlaps to a major extent to the chromosome of closely related Pantoea species. ANI values calculated for both isolates revealed two apparently new species of the Pantoea genus. Our study deciphered the identity of two bacteria producing antibiotic-like compounds, and the genomic sequence revealed they represent distinct Pantoea species.
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Pantoea , Antibacterianos/farmacologia , Israel , Pantoea/genética , Filogenia , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.
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Enzima de Conversão de Angiotensina 2 , COVID-19 , Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fator de Transcrição GATA6/genética , Humanos , Peptidil Dipeptidase A/metabolismo , Provírus/genética , SARS-CoV-2/genéticaRESUMO
The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants' mutations. We show that human sera from BriLife® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife®-acquired mutations may prove advantageous against future SARS-CoV-2 VOCs.
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BACKGROUND: The largest West African monkeypox outbreak began September 2017, in Nigeria. Four individuals traveling from Nigeria to the United Kingdom (n = 2), Israel (n = 1), and Singapore (n = 1) became the first human monkeypox cases exported from Africa, and a related nosocomial transmission event in the United Kingdom became the first confirmed human-to-human monkeypox transmission event outside of Africa. METHODS: Epidemiological and molecular data for exported and Nigerian cases were analyzed jointly to better understand the exportations in the temporal and geographic context of the outbreak. RESULTS: Isolates from all travelers and a Bayelsa case shared a most recent common ancestor and traveled to Bayelsa, Delta, or Rivers states. Genetic variation for this cluster was lower than would be expected from a random sampling of genomes from this outbreak, but data did not support direct links between travelers. CONCLUSIONS: Monophyly of exportation cases and the Bayelsa sample, along with the intermediate levels of genetic variation, suggest a small pool of related isolates is the likely source for the exported infections. This may be the result of the level of genetic variation present in monkeypox isolates circulating within the contiguous region of Bayelsa, Delta, and Rivers states, or another more restricted, yet unidentified source pool.
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Monkeypox virus , Mpox , Surtos de Doenças , Humanos , Mpox/epidemiologia , Monkeypox virus/genética , Nigéria/epidemiologia , Reino UnidoRESUMO
rVSV-Spike (rVSV-S) is a recombinant viral vaccine candidate under development to control the COVID-19 pandemic and is currently in phase II clinical trials. rVSV-S induces neutralizing antibodies and protects against SARS-CoV-2 infection in animal models. Bringing rVSV-S to clinical trials required the development of a scalable downstream process for the production of rVSV-S that can meet regulatory guidelines. The objective of this study was the development of the first downstream unit operations for cell-culture-derived rVSV-S, namely, the removal of nucleic acid contamination, the clarification and concentration of viral harvested supernatant, and buffer exchange. Retaining the infectivity of the rVSV-S during the downstream process was challenged by the shear sensitivity of the enveloped rVSV-S and its membrane protruding spike protein. Through a series of screening experiments, we evaluated and established the required endonuclease treatment conditions, filter train composition, and hollow fiber-tangential flow filtration parameters to remove large particles, reduce the load of impurities, and concentrate and exchange the buffer while retaining rVSV-S infectivity. The combined effect of the first unit operations on viral recovery and the removal of critical impurities was examined during scale-up experiments. Overall, approximately 40% of viral recovery was obtained and the regulatory requirements of less than 10 ng host cell DNA per dose were met. However, while 86-97% of the host cell proteins were removed, the regulatory acceptable HCP levels were not achieved, requiring subsequent purification and polishing steps. The results we obtained during the scale-up experiments were similar to those obtained during the screening experiments, indicating the scalability of the process. The findings of this study set the foundation for the development of a complete downstream manufacturing process, requiring subsequent purification and polishing unit operations for clinical preparations of rVSV-S.
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Vacinas contra COVID-19 , COVID-19 , Animais , Anticorpos Neutralizantes , Humanos , Pandemias , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis, as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.
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Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may influence the effectiveness of existing laboratory diagnostics. In the current study we determined whether the British (20I/501Y.V1) and South African (20H/501Y.V2) SARS-CoV-2 variants of concern are detected with an in-house S1-based antigen detection assay, analyzing spiked pools of quantitative reverse-transcription polymerase chain reaction-negative nasopharyngeal swab specimens. The assay, combining 4 monoclonal antibodies, allowed sensitive detection of both the wild type and the variants of concern, despite accumulation of several mutations in the variants' S1 region-results suggesting that this combination, targeting distinct epitopes, enables both specificity and the universality.
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COVID-19/diagnóstico , COVID-19/virologia , SARS-CoV-2/classificação , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , COVID-19/imunologia , Humanos , Mutação , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Carga ViralRESUMO
A bioterror event using an infectious bacterium may lead to catastrophic outcomes involving morbidity and mortality as well as social and psychological stress. Moreover, a bioterror event using an antibiotic resistance engineered bacterial agent may raise additional concerns. Thus, preparedness is essential to preclude and control the dissemination of the bacterial agent as well as to appropriately and promptly treat potentially exposed individuals or patients. Rates of morbidity, death, and social anxiety can be drastically reduced if the rapid delivery of antimicrobial agents for post-exposure prophylaxis and treatment is initiated as soon as possible. Availability of rapid antibiotic susceptibility tests that may provide key recommendations to targeted antibiotic treatment is mandatory, yet, such tests are only at the development stage. In this review, we describe the recently published rapid antibiotic susceptibility tests implemented on bioterror bacterial agents and discuss their assimilation in clinical and environmental samples.
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Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.