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Biology subjects

Ostrowski, M. A.

Publications and source records attributed to Ostrowski, M. A..

8 recordsLinked to original sources

Antigen stimulation drives clonal expansion of latent CD4+ T cells using a full-length HIV latency reporter

HIV persistence despite years of ART suppression poses a major barrier to cure. Using a full-length latency reporter to generate HIV-infected, transcriptionally silent CD4+ T cells in vitro, we show that cognate DC:T cell interactions drive clonal expansion of latent T cells in an antigen dependent manner and that a pro-survival state within proliferating cells is reinforced through IL-7 signaling. Interestingly, we describe a dominant role for CD28 co-stimulation in regulating robust latent T cell proliferation which was partially reversed by PD-1 blockade. Our studies show that a gradual reduction in antigenic stimulation was sufficient to induce proliferative responses without measurable proviral reactivation. Thus, the magnitude of TCR/co-stimulatory signals during cognate APC:T cell interactions are key regulators of the underlying proliferative and survival programs maintaining the latent reservoir under ART suppression. Significance StatementViral replication can be effectively suppressed by antiretroviral therapy (ART) but is not curative due to persistence of latent virus in a stable reservoir in resting CD4+ T cells. We show that antigen recognition through cell-cell interactions is an important driver of latent T cell proliferation, and that modulating TCR stimulatory signaling independently regulates proliferative, survival and proviral reactivation potential in infected T cells. Our observations show that latent T cells retain their ability to engage other immune cells to support their long-term survival under ART suppression, similar to uninfected T cells. These characteristics of latent T cell pools represent an additional hurdle to eradicating the reservoir.

immunology↗

A single-cell transposable element atlas of human cell identity

Single cell RNA sequencing (scRNA-seq) is revolutionizing the study of complex biological systems. However, most sequencing studies overlook the contribution of transposable element (TE) expression to the transcriptome. In both scRNA-seq and bulk tissue RNA sequencing (RNA-seq), quantification of TE expression is challenging due to repetitive sequence content and poorly characterized TE gene models. Here, we developed a tool and analysis pipeline for Single cell Transposable Element Locus Level Analysis of scRNA Sequencing (Stellarscope) that reassigns multi-mapped reads to specific genomic loci using an expectation-maximization algorithm. Using Stellarscope, we built an atlas of TE expression in human PBMCs. We found that locus-specific TEs delineate cell types and define new cell subsets not identified by standard mRNA expression profiles. Altogether, this study provides comprehensive insights into the influence of transposable elements in human biology.

genomics↗

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B

Safe and effective vaccines are needed to end the COVID-19 pandemic caused by SARS-CoV-2. Here we report the preclinical development of a lipid nanoparticle (LNP) formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern (VOCs), including the B.1.1.7, B.1.351 and P.1 lineages. No adverse effects were induced by PTX-COVID19-B in both mice and hamsters. These preclinical results indicate that PTX-COVID19-B is safe and effective. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 1 clinical trial ongoing (ClinicalTrials.gov number: NCT04765436). One Sentence SummaryPTX-COVID19-B is a SARS-CoV-2 mRNA vaccine that is highly immunogenic, safe, and effective in preventing SARS-CoV-2 infection in mice and hamsters and is currently being evaluated in human clinical trials.

immunology↗

Early temporal dynamics of cellular responses to SARS-CoV-2

Type I interferons (IFNs) are our first line of defence against a virus. Protein over-expression studies have suggested the ability of SARS-CoV-2 proteins to block IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wildtype SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are lacking. Here we demonstrate that SARS-CoV-2 infection induces a mild type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Our data demonstrate that SARS-CoV-2 is not adept in blocking type I IFN responses and provide support for ongoing IFN clinical trials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/158154v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@193c540org.highwire.dtl.DTLVardef@7b106forg.highwire.dtl.DTLVardef@1741cfforg.highwire.dtl.DTLVardef@1bde68_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL SUMMARY C_FIG

immunology↗

HIV Diversity Considerations in the Application of the Intact Proviral DNA Assay (IPDA)

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal ({Psi}) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4-7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

genetics↗

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.

immunology↗

Sequence, infectivity and replication kinetics of SARS-CoV-2 isolated from COVID-19 patients in Canada

SARS-CoV-2 emerged in December 2019 in Wuhan, China and has since infected over 1.5 million people, of which over 107,000 have died. As SARS-CoV-2 spreads across the planet, speculations remain about the range of human cells that can be infected by SARS-CoV-2. In this study, we report the isolation of SARS-CoV-2 from two COVID-19 patients in Toronto, Canada. We determined the genomic sequences of the two isolates and identified single nucleotide changes in representative populations of our virus stocks. More importantly, we tested a wide range of human immune cells for productive infection with SARS-CoV-2. Here we confirm that human primary peripheral blood mononuclear cells (PBMCs) are not permissive to SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor small nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine cell susceptibility and pathogenicity using in vitro and in vivo infection models.

microbiology↗

Telescope: Characterization of the retrotranscriptome by accurate estimation of transposable element expression

Characterization of Human Endogenous Retrovirus (HERV) expression within the transcriptomic landscape using RNA-seq is complicated by uncertainty in fragment assignment because of sequence similarity. We present Telescope, a computational software tool that provides accurate estimation of transposable element expression (retrotranscriptome) resolved to specific genomic locations. Telescope directly addresses uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We demonstrate the utility of our approach through single locus analysis of HERV expression in 13 ENCODE cell types. When examined at this resolution, we find that the magnitude and breadth of the retrotranscriptome can be vastly different among cell types. Furthermore, our approach is robust to differences in sequencing technology, and demonstrates that the retrotranscriptome has potential to be used for cell type identification. Telescope performs highly accurate quantification of the retrotranscriptomic landscape in RNA-seq experiments, revealing a differential complexity in the transposable element biology of complex systems not previously observed. Telescope is available at github.com/mlbendall/telescope.\n\nAuthor SummaryAlmost half of the human genome is composed of Transposable elements (TEs), but their contribution to the transcriptome, their cell-type specific expression patterns, and their role in disease remains poorly understood. Recent studies have found many elements to be actively expressed and involved in key cellular processes. For example, human endogenous retroviruses (HERVs) are reported to be involved in human embryonic stem cell differentiation. Discovering which exact HERVs are differentially expressed in RNA-seq data would be a major advance in understanding such processes. However, because HERVs have a high level of sequence similarity it is hard to identify which exact HERV is differentially expressed. To solve this problem, we developed a computer program which addressed uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We call this program, \"Telescope\". We then used Telescope to identify HERV expression in 13 well-studied cell types from the ENCODE consortium and found that different cell types could be characterized by enrichment for different HERV families, and for locus specific expression. We also showed that Telescope performed better than other methods currently used to determine TE expression. The use of this computational tool to examine new and existing RNA-seq data sets may lead to new understanding of the roles of TEs in health and disease.

genomics↗