Biology subjects

Bosinger, S. E.

Publications and source records attributed to Bosinger, S. E..

25 records · Page 2Linked to original sources

bioRxiv2021

From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/439712v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@de0663org.highwire.dtl.DTLVardef@757c30org.highwire.dtl.DTLVardef@12c4709org.highwire.dtl.DTLVardef@1ebd6d7_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryCryoEM and next generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.

immunology↗

bioRxiv2020

Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures

The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNF, CXCL8. We also identified NF-{kappa}B and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. IMPORTANCEThe current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.

microbiology↗

bioRxiv2020

Alterations in the human plasma lipidome in response to Tularemia vaccination

Tularemia is a rare but highly contagious and potentially fatal disease caused by bacteria Francisella tularensis where as few as ten inhaled organisms can lead to an infection, making it one of the most infectious microorganisms known and a potential bioweapon. To better understand the response to a live, attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated by scarification and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we identified and quantified individual lipid molecular species within representative lipid classes in plasma to characterize alterations in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-Hydroxyeicosatetraenoic acid (5-HETE), an eicosanoid produced following arachidonic acid liberation and epoxygenation, which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by Day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and several glycerophospholipids, several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

biochemistry↗

bioRxiv2019

Microbiome Stability with Chronic SIV Infection in AIDS-resistant Sooty Mangabeys

Sooty mangabeys (SMs) are a natural host species of simian immunodeficiency virus (SIV) and avoid acquired immune deficiency syndrome (AIDS) despite persistently high viral loads, making them a pivotal research model for HIV pathogenesis. Unlike pathogenic SIV infection of macaque species, or HIV infection of humans, SIV-infected SMs maintain gastrointestinal barrier integrity. Here, we characterize the gastrointestinal bacterial microbiota of SIV-infected and uninfected SMs and perform a comparative analysis of diet-matched, rhesus macaques (RM). We assessed the fecal microbiome in fifty SM and thirty RM in total, and conducted analyses of the effect of SIV-status, species, and housing. When examining indoor-outdoor and indoor-only housing in our SM cohorts, biodiversity reduction and mild phylogenetic taxonomic perturbances were present. No statistically relevant differences were seen for biodiversity richness and evenness, or phylogenetic taxonomic communities between SIV negative and positive SM cohorts. In contrast, with pathogenic early chronic SIV infections in RM a trend of alpha diversity loss and increase of beta diversity and few phyla taxonomic communities differed. Lastly, we observed lower levels of pathobiont bacterial communities in SIV-uninfected SMs relative to RMs. These data suggest that the pre-existing bacterial community structure may contribute to the divergent phenotype between SIV natural hosts and pathogenic macaque species.\n\nImportanceHuman immunodeficiency virus remains a global concern. The sooty mangabey (SM) monkey is an important biomedical research model for understanding HIV pathogenesis due to its ability to avoid AIDS disease progression despite high viremia. In people living with HIV, gastrointestinal dysbiosis towards enrichment of pathobiont communities has been frequently reported. In this study we characterized the fecal microbiota of a primate non-pathogenic SIV host, the SM, and made direct comparisons to a pathogenic SIV host species, the rhesus macaque. We observed that SMs exhibit stability of the microbiota community into chronic SIV infection, which contrasts with SIV-infected rhesus macaques, in which we observed bacterial community divergence relative to uninfected animals. Collectively, our observation of stabilization of beneficent taxa in the mucosa of AIDS-resistant primates suggests that therapeutic strategies to enrich these communities may have potential for ameliorating the gastrointestinal inflammation in people living with HIV.

immunology↗

bioRxiv2019

Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and Topoisomerase Inhibitors

Abstract Abstract Importance Introduction Results Discussion Materials and Methods References Breast cancer is the second-leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast cancer. Mammalian orthoreovirus (reovirus) selectively infects and kills transformed cells and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent against several cancers. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and if reovirus-induced cytotoxicity of breast cancer cells can be enhanced by modu ...

microbiology↗

bioRxiv2018

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-{beta} mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.\n\nIMPORTANCEWest Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

microbiology↗

bioRxiv2018

Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance

The observation that humans can produce broadly neutralizing antibodies (bnAbs) against HIV-1 has generated enthusiasm about the potential for a bnAb vaccine against HIV-1. Conventional immunization strategies will likely be insufficient for the development of a bnAb HIV vaccine and vaccines to other difficult pathogens, due to the significant immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. Using longitudinal lymph node fine needle aspirates, we found that two independent methods of slow delivery immunization of rhesus macaques (RM) resulted in larger GCs, more robust and sustained GC Tfh cell responses, and GC B cells with improved Env-binding, which correlated with the development of ~20 to 30-fold higher titers of tier 2 HIV-1 nAbs. Using a new RM genomic immunoglobulin loci reference sequence, we identified differential IgV gene usage between slow delivery immunized and conventional bolus immunized animals. The most immunodominant IgV gene used by conventionally immunized animals was associated with many GC B cell lineages. Ab mapping of those GC B cell specificities demonstrated targeting of an immunodominant non-neutralizing trimer base epitope, while that response was muted in slow delivery immunized animals. Thus, alternative immunization strategies appear to enhance nAb development by altering GCs and modulating immunodominance of non-neutralizing epitopes.

immunology↗