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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). We identified several host factors critical for SARS-CoV-2 infection. Comparative analysis of the different VOCs revealed 2 host factors as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6 as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and is critical for SARS-CoV-2 cell entry. 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.
Published: Nature communications, 25 April 2022
The paper describes the optimization of the production of the VSV-ΔG-spike vaccine in Vero cells using the Ambr15 system. During optimization, critical parameters were tested. Those parameters included: cell densities; the multiplicity of infection; virus production temperature; medium addition and medium exchange; and supplementation of glucose in the virus production step. Virus production temperature, medium addition, and medium exchange were all found to significantly influence the yield. The optimized parameters were tested in the BioBLU 5p bioreactors production process and those that were found to contribute to the vaccine yield were integrated into the final process. The findings of this study demonstrate that an Ambr15 system is an effective tool for bioprocess optimization of vaccine production using macrocarriers and that the combination of production temperature, rate of medium addition, and medium exchange significantly improved virus yield.
Published: Biotechnology & Bioengineering, 23 of March, 2022
Transcriptome analysis of lungs in a mice model of severe COVID-19, predicts clinical manifestations that are consistent with the developing disease in humans following infection with the SARS-CoV2 virus, thereby establishing the suitability of this unique animal model for the study of COVID-19 disease.
Published: Front.Virol., 21 March 2022
The SARS-CoV-2 Omicron strain emergence raised concerns that its enhanced infectivity is partly due to altered spread/contamination modalities. Herein, we sampled high-contact surfaces and air in close proximity to patients verified as infected with Omicron strain, original or Alpha strains. Cumulatively, for all three strains, viral-RNA was detected in 90/168 surfaces and 6/49 air-samples. No infective virus was identified. No significant differences in prevalence were found between strains. This preliminary work suggests that Omicron’s increased transmissibility doesn't result from acquiring airborne infectivity, higher environmental contamination or better resilience on surfaces.
Published: The International Journal of Infectious Diseases, 5 March 2022
The paper describes a mass spectrometric (MS) spike antigen quantification method, applied to VSV-ΔG-spike based vaccine (BriLife). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Proof of concept of this method with two different sample preparations is shown for complex matrix samples. Total spike levels were correlated with results from activity assays, and ranged between 0.3-0.5 µg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.
Published: The Journal of Virological Methods, 22 February 2022
Emerging SARS-CoV-2 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, unlike a significant drop observed in other vaccines. 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.
Published: Vaccines, 14 of February , 2022
Cellular immunity involving the activity of T cells is fundamental for preventing the development of severe COVID-19 condition following SARS-CoV-2 infection. Our newly-developed tools enabled monitoring T cell reactivity against the spike protein. Using peripheral cells collected from individual vaccinated against SARS-CoV-2, we compared the response to ancestral and Omicron spike and found a comparable level of response. These data is highly relevant for risk assessment in response to emerging variants of concern.
Published: Viruses, 8 of February, 2022
Here, we present a reliable, specifc, and rapid method for the identifcation of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC–MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while signifcantly reducing background noise interrupting with LC–MS analysis. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated and successfully applied to clinical NP samples.
Published: Analytical and Bioanalytical Chemistry, 4 January 2022
In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of Poly(I:C), against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate, and transient upregulation of innate immunity-related genes in lungs. This effect was accompanied by viral load reduction, lung and brain cytokine storms prevention, resulted in mice survival.
Published: Viruses,19 January 2022
This article describes novel method that enables in-line and fast monitoring of purification processes in the downstream stages. The method is using automatic chemical analayzer. The main importance of this method is the continuous tracking after the removing of small molecules, mainly metabolites and substrates, that produced or were add to the purified solution in the upstream stage. Using that method enable the tracking the diafiltration progress especially in process where the pH and conductivity parameters are similar. The number of buffer exchange in the diafiltration stage in the production process of the rVSV-ΔG-Spike vaccine were determined using that method. Moreover, that article describes the use of the automatic chemical analyzer for fast tracking after the removing of LDH protein in the purification stages of the rVSV-ΔG-Spike vaccine downstream process
Published: BioTec, 3 November 2021
A review describing the principles for designing an optimal mRNA lipid nanoparticle vaccine
Published: Current Opinion in Biotechnology (COBIOT), 26 October 2021
The study reports the process development of small scale, pilot scale and large scale of endonuclease digestion of hc-DNA, clarification by filtration and ultrafiltration for production of rVSV-ΔG-Spike vaccine against SARS-CoV-2. The main pert of the article deals with the development of efficient ultrafiltration process within maintaining high viral yield. At the ultrafiltration studies different filter types, shear rates, working pressure and the ratio solution/surface area. Base on that study large scale process was developed. The large-scale process was used to production of rVSV-ΔG-Spike vaccine for phase I/II clinical experiments that are described in that paper.
Published: Vaccine, 22 October 2021
The study reports the process development of highly efficient chromatographic step for the purification of rVSV-ΔG-spike vaccine against SARS-CoV-2. Several chromatographic methods were evaluated including membrane adsorbers and packed-bed-ion-exchange resins. It was found that the resin captocore 700, a porous resin (700KD cutoff) which charged with positive charge inside the porous (strong anionic exchanger), is the most efficient for the vaccine purification. In the chromatographic process the viruses that their molecular weight in larger that 700KD don’t penetrate to the resin porous and elute in the flowthrough. On the other hand, the anionic contaminates that includes most of the host cell proteins, Cellular DNA, DNase and etc. are bound to the resin and elute in the regeneration stage. The high efficiency of the chromatographic purification using the captocore 700 resin enables the implementation of the chromatographic stage at the earliest stages of the downstream process in the vaccine manufacturing.
Published: BioTech, 12 October 2021
We examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV infected mice to developing severe and fatal disease upon coinfection with SARS-CoV-2 two days. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic.
Published: Nature communications, 5 October, 2021
A wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha, and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants.
Published: Cell Reports, 21 of August 2021
We report a new methodology for the detection of unknown VOCs in breath. A successful demonstration of this method was conducted in a pilot study (n = 21) that took place in a closed hospital ward (Covid-19 ward) with the discovery of four potential markers.
Mice are not susceptible to SARS infection, and cannot serve as a model for COVID-19. Recently, we implemented a novel method for expressing the SARS receptor in the lungs of immunologically humanized mice and consequently to sensitize them to SARS infection. The method enables evaluation of various anti COVID-19 therapies.
Published: Pathogens, 26 of July, 2021
A hybrid stochastic mathematical model was formulated in order to investigate the pre-symptomatic SARS-CoV-2 infection phenomenon. It describes discrete human actions as well as continuous virus decay processes which occur in a household environment. The dominant driver of pre-symptomatic transmission was found to be direct contact route (64.5% of total viral dose). This route occurs when infected people touch their own faces and then touch other individuals. The relative contribution of other routes to the total viral dose was 26% for indirect contact via fomites (frequently touched objects) and 9.5% for airborne droplet nuclei. The contribution of indirect contact mediated by environmental surfaces was negligible. Wearing masks and avoiding physical contact are an effective prevention strategy.
Published: Sci Rep, 14 of July 2021
Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing COVID-19 outbreaks. Here we present LAMP-Seq, a modified highly scalable method. Swab samples are barcoded and amplified in a single heat step, and pooled products are analyzed by sequencing. Clinical validation demonstrated 100% sensitivity and 99.7% specificity in individuals with Ct33. With less then 24h to result, low cost and little infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.
Published: Nature Biotechnology, 29 June 2021
Human leukocyte antigen (HLA)-bound-viral antigens can be selectively recognized by T cells to mount anti-viral T cell immunity. In a multi-lab collaboration study, between scientists from the IIBR, with the laboratory of prof. Yardena Samuels at the Weizmann institute of science and scientists at NKI (Netherland), Tissue typing unit at Hadassah medical center, Stanford university, utilizing HLA-peptidomics, we were able to identify SARS-CoV-2- derived peptides presented by highly prevalent HLA Class-I (HLA-I) molecules using infected cells as well as overexpression of SARS-CoV-2 genes. We found 26 HLA-I peptides and 36 HLA class-II (HLA-II) peptides. Among the identified peptides few were shown to be immunogenic. These results may aid the development of the next generation of SARS-CoV-2 vaccines based on viral-specific-presented antigens that span several of the viral genes
Publishes: Cell Reports, 29 of June 2021
A broadly ranging, simple and rapid method for counting cells on carriers is described and exemplified using Fibra-Cel macro carriers. Authors show employment of the method as part of the development of SARS-CoV-2 vaccine to track cell adsorption, cell growth and viability on carriers. The method provides comprehensive details necessary for process control of viral vaccine production in cells and can be easily implemented in any cell-based processes and other unique platforms for measuring growth of encapsulated cells.
Published: Biotechnology & Bioengineering, 10 of June, 2021
An in vitro binding assay between the receptor binding domain (RBD) of SARS-CoV-2 and ACE2 was developed, and applied to identify inhibitors by screening of two compound libraries for COVID-19 drug repurposing. Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition. A plaque reduction assay in Vero E6 cells infected with VSV-SARS-CoV-2-S, a SARS-CoV-2 surrogate virus, confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD.
Published: Molecules, 27 of May 2021
Here we report the isolation, characterization, and recombinant production of 12 neutralizing human mAbs, targeting three distinct epitopes on the spike N-terminal domain of the virus. Neutralization mechanism of these antibodies involves receptors other than the canonical hACE2 on target cells, relying both on amino acid and N-glycan epitope recognition, suggesting alternative viral cellular portals. Two selected mAbs demonstrated full protection of K18-hACE2 transgenic mice when administered at low doses and late postexposure, demonstrating the high potential of the mAbs for therapy of SARSCoV-2 infection.
Published: iScience. 21 of May 2021
Coronaviruses developed varied mechanisms to repress host mRNA translation to allow the translation of viral mRNAs and concomitantly block the cellular innate immune response. Yet, comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. In a collaboration study between scientists at the IIBR and the Weizmann Institute of Science, we combined RNA-sequencing, ribosome profiling and metabolic labeling of newly synthesized RNA, to comprehensively define the mechanisms that are utilized by SARS-CoV-2 to shutoff cellular protein synthesis. We show that infection leads to accelerated degradation of cytosolic cellular mRNAs which facilitates viral takeover of the mRNA pool in infected cells. Moreover, we reveal that the translation of transcripts whose expression is induced in response to infection, including innate immune genes, is impaired. We demonstrate this impairment is likely mediated by inhibition of nuclear mRNA export, preventing newly transcribed cellular mRNAs from accessing ribosomes. Overall, our results uncover the multipronged strategy employed by SARS-CoV-2 to commandeer the translation machinery and to suppress host defenses.
Published: Nature, 21 Of May 2021
The current study documents the successful identification and recombinant production of a set of anti-RBD and anti-NTD-specific mAbs isolated by a single-cell sorting strategy of lymphocytes collected from rabbits immunized with different spike-derived antigens. These antibodies were characterized as having high specificity and affinity profiles. Anti-RBD antibodies showed in vitro neutralizing potency against SARS-CoV-2 directed to a novel epitope within the RBD. These antibodies may represent the basis for future development of immunodiagnostics and immunotherapy.
Publishe: Viruses, 26 of March, 2021
In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike’s S1 subunit. The assay’s performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results.
Published: Analytical and Bioanalytical Chemistry, 25 of March, 2021
In the present study, we characterized and further evaluated the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. 75% of the untreated mice succumbed 6-9 days post-infection while administration of the MD65 antibody as late as 3 days after exposure, rescued all infected animals. The data unprecedentedly demonstrate, the therapeutic value of human monoclonal antibodies as a life-saving treatment of severe COVID-19 infection.
Published: Nature Communications, 11 February 2021
A development of a novel assay for SARS-CoV-2 identification using LC-MS/MS analysis. A multi-step procedure for the rational down-selection of a set of markers has leaded to the discovery of six SARS-CoV-2 specific and sensitive markers, enabling the reliable identification of the virus. A rapid and simple assay was developed, successfully applied to clinical nasopharyngeal samples. The assay may potentially serve as a complementary approach for SARS-CoV-2 identification.
Published: American chemical Society, 26 January 2021
We report the genome sequences and the identification of genetic variations in eight clinical samples of SARS-CoV-2 collected from individuals from elderly and infirm persons in Israel. The variant calling process revealed a total of 51 substitutions that were previously documented, and one non-synonymous replacement, that has not been documented before, and is located in the nucleocapsid protein. Mapping and identification of new mutations may contribute to a better understanding of the viral factors related to the clinical outcomes of the disease.
Published: Microbiology Resource Announcements, 7 January 2021
A vaccine against SARS-CoV-2 based on a recombinant virus expressing the SARS-CoV-2 spike protein was developed. The efficacy of the vaccine was demonstrated by a rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Moreover, a single dose vaccination was able to protect hamsters against SARS-CoV-2.
Published: Natune communications, 16 December 2020
This is our vaccine's story from research to production
An important milestone was completed.
To estimate the risk of SARS-CoV-2 transmission to humans by surface contact, we examined the stability of the virus on variety of surfaces, typical of urban environment or patient environment. We used PCR to detect the virus, tissue culture based growth test to determine virus viability and ability to infect new host. At room temperature, SARS-CoV-2 gradually loss its viability and decay completely at day 4. The decay rate increases at higher temperatures. About 50% of the samples from the patient surrounding were PCR positive while none of them was viable (0/97), therefore, aerosol or indirect transmission from inanimate surfaces around hospitalized or quarantined COVID-19 patients is not supported by the data presented in this study. Fomite transmission may still be a possibility with heavily contaminated surfaces around patients during their most contagious stages of infection, and in closed and crowded environments.
Published: Clinical Microbiology and Infection, 10 September 2020
A research collaboration between the Israel Institute for Biological Research, in Ness Ziona and the Weizmann Institute of Science, Israel uncovered the coding capacity of the SARS CoV-2 virus. The researchers applied an innovative method of tracking translating ribosomes on their RNA template and converting the data into a translational-map of all the proteins being translated in the infected cells. Using this innovative method, resulted in the identification of 23 new short protein (peptide) sequences in the SARS-CoV-2 genome that can contribute in a regulatory function, but four were full-fledged proteins and might serve as new tools in diagnostic, prevention or treatment of COVID-19.
Published: Nature 9 September 2020
IIBR scientists have isolated and characterized a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor-binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined treatment of COVID-19 patients or for prophylaxis immunization.
Published: Nature Comminications, 27 August 2020
Selected excerpts from IIBR’s Director General, Prof. Shmuel C. Shapira’s session to the Science and Technology Committee at The Knesset
SARS-Cov-2 genetic identification is based on viral RNA extraction prior to RT -qPCR assay. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified up to 70% pf positive clinical specimens.
Published: International Journal of Infectious Diseases, 5 August 2020
We announce the genome sequences of two strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated in Israel, one imported by a traveler who returned from Japan and the second strain collected from a patient infected by a traveler returning from Italy. The sequences obtained are valuable as early manifestations for future follow-up of the local spread of the virus in Israel.
Published: Microbiology Resource Announcements, 9 July 2020
We tested the efficacy of face shields as a substitution for medical masks in blocking cough-like generated spray. Our results imply that blocking efficacy of face shields is similar, and for some parts of the face, even higher, than the efficacy of medical masks. This is even truer for fine particles, which are blocked much more efficiently by a shield. Considering other advantages of shields, public health policy makers may consider the usage of face shields as an alternative to medical masks for the general population.
A new study examined the antiviral effect of two specific inhibitors of GlucosylCeramide synthase (GCS): (1). an analogue of the FDA-approved drug Cerdelga®, (2) an analogue of venglustat which is currently in phase III clinical trials. Our findings show that both inhibitors inhibit the replication of four different enveloped RNA viruses of different genus, organ-target and transmission route, including SARS-Cov-2. The fact that these inhibitors have an antiviral effect on viruses of four different families, suggests a key role of the glycosphingolipid synthesis pathway in viral infection. Targeting host proteins or pathways utilized by multiple viruses is less prone to the development of resistance to the drug through mutations.
It is not clear what mechanism enables infection by pre-symptomatic COVID-19 individuals, especially in indoors environments. IIBR scientists have answered this question by formulating an evidence-based mathematical model. The major infection mechanism was found to be physical person-to-person contact. Infection may be induced by touching fomites (small, frequently touched surfaces) but it is highly unlikely to be infected by airborne route. Both contact routes are induced by pre-symptomatic individuals touching their facial membranes with their hand and then touching other people or fomites. The conclusion is that the social distance directive should be clarified in order to emphasize the importance of refrain from any physical contact.
IIBR will continue to characterize these antibodies and others and a biological manufacture would be sought to mass produce it for clinical use.
A joint ongoing study led by IIBR researchers and physicians from Laniado and Assuta Ashdod hospitals tested the viability of corona virus in patient‘s environment surfaces. The study reveals low probability of being infected due to surface contact.
IIBR, using its Biology division's well-established platform for antibody engineering, have managed to isolate a monoclonal antibody against the virus, from blood samples that were obtain from severe COVID19 patients. It was also demonstrated that this antibody was able to effectively neutralize the virus. Parallel efforts are being made worldwide. Some have managed to isolate antibodies that neutralize other closely related viruses and even anti-SARS-CoV-2 monoclonal antibodies, but IIBR is the first to demonstrate that the antibody that was isolated from COVID19 patient can indeed neutralize this pathogen. IIBR will continue to characterize this antibody and others and a biological manufacture would be sought to mass produce it for clinical use.
IIBR Director General, Professor Shmuel Shapira, informed Prime Minister Benjamin Netanyahu of "significant progress" in designing a vaccine prototype and that the institute "is now preparing a model for commencing an animal trial".
The Israel institute for biological research is assigned by the Israeli Prime mister, Mr. Benjamin Netanyahu, to produce a vaccine and antibody for the Novel Coronavirus.