Biology subjects

Cook, A.

Publications and source records attributed to Cook, A..

53 records · Page 3Linked to original sources

bioRxiv2021

Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 {micro}g of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.{beta} (heterologous), which encompasses the spike sequence of the B.1.351 (beta or {beta}) variant. Reciprocal ID50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the {beta} variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost {beta}-specific reciprocal ID50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the {beta} variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations. One-sentence summarymRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.

immunology↗

bioRxiv2021

A modular protein subunit vaccine candidate produced in yeast confers protection against SARS-CoV-2 in non-human primates

Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>104) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log10 reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.

immunology↗

bioRxiv2021

Evaluation of mRNA-1273 against SARS-CoV-2 B.1.351 Infection in Nonhuman Primates

BackgroundVaccine efficacy against the B.1.351 variant following mRNA-1273 vaccination in humans has not been determined. Nonhuman primates (NHP) are a useful model for demonstrating whether mRNA-1273 mediates protection against B.1.351. MethodsNonhuman primates received 30 or 100 {micro}g of mRNA-1273 as a prime-boost vaccine at 0 and 4 weeks, a single immunization of 30 {micro}g at week 0, or no vaccine. Antibody and T cell responses were assessed in blood, bronchioalveolar lavages (BAL), and nasal washes. Viral replication in BAL and nasal swabs were determined by qRT-PCR for sgRNA, and histopathology and viral antigen quantification were performed on lung tissue post-challenge. ResultsEight weeks post-boost, 100 {micro}g x2 of mRNA-1273 induced reciprocal ID50 neutralizing geometric mean titers against live SARS-CoV-2 D614G and B.1.351 of 3300 and 240, respectively, and 430 and 84 for the 30 {micro}g x2 group. There were no detectable neutralizing antibodies against B.1351 after the single immunization of 30 {micro}g. On day 2 following B.1.351 challenge, sgRNA in BAL was undetectable in 6 of 8 NHP that received 100 {micro}g x2 of mRNA-1273, and there was a [~]2-log reduction in sgRNA in NHP that received two doses of 30 {micro}g compared to controls. In nasal swabs, there was a 1-log10 reduction observed in the 100 {micro}g x2 group. There was limited inflammation or viral antigen in lungs of vaccinated NHP post-challenge. ConclusionsImmunization with two doses of mRNA-1273 achieves effective immunity that rapidly controls lower and upper airway viral replication against the B.1.351 variant in NHP.

immunology↗

bioRxiv2021

Immune Correlates of Protection by mRNA-1273 Immunization against SARS-CoV-2 Infection in Nonhuman Primates

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 - 100 g of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naive hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP. One-Sentence SummarymRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.

immunology↗

bioRxiv2021

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques

Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 {micro}g RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only [~]2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. Significance StatementThe emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.

immunology↗

bioRxiv2021

Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 {micro}g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFNs potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. One-Sentence SummaryA SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

immunology↗

bioRxiv2021

Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3x106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

immunology↗

bioRxiv2021

Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. In this study, we evaluated the immunogenicity and protective efficacy of reduced doses of Ad26.COV2.S. 30 rhesus macaques were immunized once with 1x1011, 5x1010, 1.125x1010, or 2x109 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes. Vaccine doses as low as 2x109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125x1010 vp were required for protection in nasal swabs. Activated memory B cells as well as binding and neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show evidence of virologic, immunologic, histopathologic, or clinical enhancement of disease compared with sham controls. These data demonstrate that a single immunization with a relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques. Moreover, our findings show that a higher vaccine dose may be required for protection in the upper respiratory tract compared with the lower respiratory tract.

immunology↗

bioRxiv2021

A targeted and tuneable DNA damage tool using CRISPR/Cas9.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

cell biology↗

bioRxiv2020

A Tunable, Simplified Model for Biological Latch Mediated Spring Actuated Systems

We develop a model of latch-mediated spring actuated (LaMSA) systems relevant to comparative biomechanics and bioinspired design. The model contains five components: two motors (muscles), a spring, a latch, and a load mass. One motor loads the spring to store elastic energy and the second motor subsequently removes the latch, which releases the spring and causes movement of the load mass. We develop open-source software to accompany the model, which provides an extensible framework for simulating LaMSA systems. Output from the simulation includes information from the loading and release phases of motion, which can be used to calculate kinematic performance metrics that are important for biomechanical function. In parallel, we simulate a comparable, directly actuated system that uses the same motor and mass combinations as the LaMSA simulations. By rapidly iterating through biologically relevant input parameters to the model, simulated kinematic performance differences between LaMSA and directly actuated systems can be used to explore the evolutionary dynamics of biological LaMSA systems and uncover design principles for bioinspired LaMSA systems. As proof of principle of this concept, we compare a LaMSA simulation to a directly actuated simulation that includes a either Hill-type force-velocity trade-off or muscle activation dynamics, or both. For the biologically-relevant range of parameters explored, we find that the muscle force-velocity trade-off and muscle activation have similar effects on directly actuated performance. Including both of these dynamic muscle properties increases the accelerated mass range where a LaMSA system outperforms a directly actuated one.

biophysics↗

bioRxiv2020

Investigating the role of APOE: limitations of human pluripotent stem cell-derived cerebral organoids

Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimers disease, with the presence of APOE-{varepsilon}4 associated with increased risk of developing Alzheimers disease. Here, we reprogrammed human fibroblasts from individuals with different APOE-{varepsilon} genotypes into induced pluripotent stem cells, and generated isogenic lines with different APOE profiles. We then differentiated these into cerebral organoids for six months and assessed the suitability of this in vitro system to measure APOE, {beta} amyloid, and Tau phosphorylation levels. We identified intra- and inter-variabilities in the organoids cell composition. Using the CRISPR-edited APOE isogenic lines, we observed more homogenous cerebral organoids, and similar levels of APOE, {beta} amyloid, and Tau between the isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-{varepsilon}4/{varepsilon}4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-{varepsilon} status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the batch-to-batch and cell-line-to-cell-line variabilities need to be considered when using cerebral organoids.

neuroscience↗

bioRxiv2020

REGN-COV2 antibody cocktail prevents and treats SARS-CoV-2 infection in rhesus macaques and hamsters

An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.

microbiology↗

bioRxiv2020

Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. Here, we assess immunogenicity and anamnestic protective efficacy in rhesus macaques of the intradermal (ID)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800. INO-4800 is an ID-delivered DNA vaccine currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and neutralizing antibody responses against both the D614 and G614 SARS-CoV-2 spike proteins. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T and B cell responses. These responses were associated with lower viral loads in the lung and with faster nasal clearance of virus. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against COVID-19 disease.

immunology↗

bioRxiv2020

SALL4 controls cell fate in response to DNA base composition

SO_SCPLOWUMMARYC_SCPLOWMammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4 which contains multiple zinc-fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely up-regulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4-null cells, including precocious differentiation of embryonic stem cells and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by down-regulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive by-product of genome evolution, but constitutes a signal that aids control of cell fate. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/179481v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1ec4eb9org.highwire.dtl.DTLVardef@1246a36org.highwire.dtl.DTLVardef@1b9134dorg.highwire.dtl.DTLVardef@1a24904_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

PRP4K is a haploinsufficient tumour suppressor negatively regulated during epithelial-to-mesenchymal transition

The pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential gene. However, reduced PRP4K expression is associated with aggressive breast and ovarian cancer phenotypes including taxane therapy resistance, increased cell migration and invasion in vitro and cancer metastasis in mice; results consistent with PRP4K being a haploinsufficient tumour suppressor. Increased cell migration and invasion is associated with epithelial-to-mesenchymal transition (EMT), but how reduced PRP4K levels affect normal epithelial cell migration or EMT has not been studied. Depletion of PRP4K by small hairpin RNA (shRNA) in non-transformed mammary epithelial cell lines (MCF10A, HMLE) reduced or had no effect on 2D migration in the scratch assay but resulted in greater invasive potential in 3D transwell assays. Depletion of PRP4K in mesenchymal triple negative breast cancer cells (MDA-MB-231) resulted in both enhanced 2D migration and 3D invasion, with 3D invasion correlated with higher fibronectin levels in both MDA-MB-231 and MCF10A cells and without changes in E-cadherin. Induction of EMT in MCF10A cells, by treatment with WNT-5a and TGF-{beta}1, or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, resulted in significantly reduced PRP4K expression. Mechanistically, induction of EMT by WNT-5a/TGF-{beta}1 reduced PRP4K transcript levels, whereas eIF3e depletion led to reduced PRP4K translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, both of which are reversed by overexpression of exogenous PRP4K. Thus, PRP4K is a haploinsufficient tumour suppressor negatively regulated by EMT, that when depleted in normal mammary cells can increase cell invasion without inducing full EMT.

cancer biology↗

bioRxiv2018

Immune regeneration in irradiated mice is not impaired by the absence of DPP9 enzymatic activity

The ubiquitous intracellular protease dipeptidyl peptidase 9 (DPP9) has roles in antigen presentation and B cell signaling. To investigate the importance of DPP9 in immune regeneration, primary and secondary chimeric mice were created in irradiated recipients using fetal liver cells and adult bone marrow cells, respectively, using wild-type (WT) and DPP9 gene-knockin (DPP9S729A) enzyme-inactive mice. Immune cell reconstitution was assessed at 6 and 16 weeks post-transplant. Primary chimeric mice successfully regenerated neutrophils, natural killer, T and B cells, irrespective of donor cell genotype. There were no significant differences in total myeloid cell or neutrophil numbers between DPP9-WT and DPP9S729A-reconstituted mice. There were fewer B cells in the DPP9S729A-origin compared to DPP9 WT-origin mice in primary chimeric mice. In secondary chimeric mice, cells of DPP9S729A-origin cells displayed enhanced engraftment compared to WT. However, we observed no differences in myeloid or lymphoid lineage reconstitution between WT and DPP9S729A donors, indicating that hematopoietic stem cell (HSC) engraftment and self-renewal is not diminished by the absence of DPP9 enzymatic activity. This is the first report on transplantation of bone marrow cells that lack DPP9 enzymatic activity.

immunology↗

bioRxiv2017

A Reagentless Biosensor For mRNA: A New Tool To Study Transcription

Gene expression, catalysed by RNA polymerases, is one of the most fundamental processes in living cells. Yet, the means to study their activity are currently limited. The majority of methods to quantify mRNA are based upon initial purification of the nucleic acid. This leads to experimental inaccuracies and loss of product. Here, we describe the use of a reagentless mRNA fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, SSB showed similar binding properties to mRNA, to that of its native substrate, ssDNA. Furthermore, fluorescently labelled MDCC-SSB gave the same fluorescence response with both ssDNA and ssRNA, in a concentration dependent manner. When directly compared to RT-qPCR, we found the biosensor to be more reproducible with no product lost through purification. Therefore, the MDCC-SSB is a novel tool for comparative measurement of mRNA yield following in vitro transcription.

biochemistry↗