Direct binding and internalization of diverse extracellular nucleic acid species through the collagenous domain of class A scavenger receptors.

Nucleic acids are potential pathogen-associated or danger-associated molecular patterns that modulate immune responses and the development of autoimmune disorders. Class A scavenger receptors (SR-As) are a diverse group of pattern recognition receptors that recognize a variety of polyanionic ligands including nucleic acids. While SR-As are important for the recognition and internalization of extracellular dsRNA, little is known about extracellular DNA, despite its association with chronic infections and autoimmune disorders. In this study, we investigated the specificity of and requirement for SR-As in binding and internalizing different species, sequences and lengths of nucleic acids. We purified recombinant coiled-coil/collagenous and scavenger receptor cysteine-rich (SRCR) domains that have been implicated as potential ligand-binding domains. We detected a direct interaction of RNA and DNA species with the coiled-coil/collagenous domain, but not the SRCR domain. Despite the presence of additional surface receptors that bind nucleic acids, SR-As were found to be sufficient for nucleic acid binding and uptake in A549 human lung epithelial cells. Moreover, these findings suggest that the coiled-coil/collagenous domain of SR-As is sufficient to bind nucleic acids independent of species, sequence or length.

Class A Scavenger Receptor-Mediated Double-Stranded RNA Internalization Is Independent of Innate Antiviral Signaling and Does Not Require Phosphatidylinositol 3-Kinase Activity.

dsRNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and after release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As) mediate dsRNA entry, it is unknown whether they contribute to signaling beyond ligand internalization. In this study, we investigated whether SR-As contribute to innate immune signaling independent of the classic TLR and retinoic acid-inducible gene-I-like receptor (RLR) pathways. We generated a stable A549 human epithelial cell line with inducible expression of the hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IFN-ß promoter stimulator 1, adaptors for TLR3 and the RLRs, respectively. Cells expressing NS3/4A and TLR3/MyD88/IFN-ß promoter stimulator 1(-/-) mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I IFN. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, whereas downstream signaling leading to IFN production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling.

The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite.

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.

Depth filtration for clarification of intensified lentiviral vector suspension cell culture.

Depth filtration significantly impacts efficiency of lentiviral (LV) vector purification process. However, it is often deprioritized in the overall scope of viral vector manufacturing process optimization. The demand for LV vectors has increased with the rise in disease indications, making it crucial to improve current manufacturing processes. Upstream bioreactor process intensification has enabled cell densities of over 107 viable cells/mL, creating challenges for harvest unit operations. The larger size of LV vectors and their physiochemical similarity to host cell-DNA (HC-DNA) and poor clarification performance causes significant challenges for the subsequent chromatography-based purifications. As a result, a robust and scalable harvest of LV process is needed, especially for LV in vivo therapeutic quality needs. In this study, we systematically evaluated the overlooked yet important issue of depth filtration systems to improve enveloped LV functional vector recovery. We found that an established depth filtration system in process A that provided 94% (n = 6) LV functional recovery could not be translated to intensified Process B cell culture. Hence, the depth filtration process became a bottleneck for the purification performance in an intensified process. We demonstrated an improvement in LV functional vector recovery from 34% to 82% via filter train optimization for an intensified suspension cell culture system (>107 cells/mL with higher titer), while still maintaining a loading throughput of ≥82 L/m2 and turbidity ≤20 NTU. It was demonstrated that the two or three-stage depth filtration scheme is scalable and more suitable for high cell density culture for large scale for LV manufacturing process.

Next-Generation Sequencing: A Promising Tool for Vaccines and Other Biological Products.

Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), is a commonly used term to represent a set of DNA sequencing technologies that have been in use for almost two decades [...].

T-cell responses following Natural Influenza Infection or Vaccination in Solid Organ Transplant Recipients.

Little is known about cell-mediated immune responses to natural influenza infection in solid organ transplant (SOT) patients. The aim of our study was to evaluate the CD4+ and CD8+ responses to influenza A and B infection in a cohort of SOT patients. We collected peripheral blood mononuclear cells at influenza diagnosis and four weeks later from 31 SOT patients during the 2017-2018 influenza season. Infection-elicited influenza-specific CD4+ and CD8+ T-cell responses were measured using flow cytometry and intracellular cytokine staining and compared to responses following influenza vaccine in SOT patients. Natural infection was associated with a significant increase in CD4+ T-cell responses. For example, polyfunctional cells increased from 21 to 782 and from 193 to 1436 cells per 106 CD4+ T-cells among influenza A/H3N2 and B-infected patients (p = 0.006 and 0.004 respectively). Moreover, infection-elicited CD4+ responses were superior than vaccine-elicited responses for influenza A/H1N1 (931 vs 1; p = 0.026), A/H3N2 (647 vs 1; p = 0.041) and B (619 vs 1; p = 0.004). Natural influenza infection triggers a significant increase in CD4+ T-cell responses in SOT patients. Infection elicits significantly stronger CD4+ responses compared to the influenza vaccine and thereby likely elicits better protection against reinfection.

Incidence, Outcomes, and Long-term Immune Response to Tuberculosis in Organ Transplant Recipients.

BACKGROUND: Tuberculosis (TB) is a significant opportunistic infection in solid organ transplant recipients (SOTR). There are limited data on TB incidence in transplantation from low prevalence countries as well as on long-term TB-specific immune responses. METHODS: We performed a single-center retrospective review of SOTR diagnosed with active TB between 2000 and 2015 and further contacted the available patients for a study of long-term T-cell responses using an interferon-gamma (IFN-γ) release assay and a flow cytometry-based assay. RESULTS: We identified 31 SOTR with active TB for an incidence of 62 cases/100 000 patient-years. Nineteen (61.3%) of 31 patients were diagnosed within the first year after transplant. Nineteen (61.3%) were born in countries with high TB prevalence and disseminated disease occurred in 22.6%. No patient had been screened for latent TB infection pretransplant. The majority of patients received isoniazid and a rifamycin as part of multidrug regimen. In addition, 13 (44.8%) of 29 patients received quinolones. One-year mortality in this population was 19.4%. Eight patients were available for long-term immune responses. Of these, all had detectable IFN-γ response by IFN-γ release assay testing and 7 of 8 had detectable TB-specific T cells, primarily central and effector T-cell responses in the CD4 compartment and terminally differentiated T cells in the CD8 compartment. CONCLUSIONS: TB has high incidence in SOTR even in low-prevalence regions but especially targets patients who originated from TB-endemic countries. Long-term TB-specific T-cell responses were found in the majority of patients.

Extracellular dsRNA: its function and mechanism of cellular uptake.

Double-stranded RNA (dsRNA) is arguably the most potent viral trigger of innate immune signaling. Its activity has been recognized for over 5 decades, first as a toxin, then as a central component of the interferon system, as an efficient activator of antiviral responses and an immunomodulator for therapeutic applications. Nucleic acid sensing is the main basis for antiviral defense systems throughout the diverse forms of life from bacteria to plants and animals. Pattern recognition receptors of the host defense system not only sense viral dsRNA as a pathogen-associated molecular pattern in infected cells, but also recognize circulating endogenous dsRNA, a nonmicrobial signal, as a danger-associated molecular pattern, often leading to autoimmunity. Despite the effects of extracellular viral and host dsRNA associated with infection and autoimmunity, respectively, the understanding of cellular mechanisms for its recognition and uptake has only been appreciated in recent years. This review presents an overview of this unique form of nucleic acid, addressing its roles in infection, autoimmunity, and host sensing mechanisms. The goal of this review is to highlight the novel findings with a focus on extracellular recognition and uptake by the cell.

Post-transcriptional regulation of the U3 small nucleolar RNA.

A high copy shuttle vector was used to express a "tagged" U3 small nucleolar RNA (snoRNA) gene in Schizosaccharomyces pombe to examine regulatory responses to a high gene dosage. RNA analyses utilizing reverse transcription-PCR amplification and restriction fragment length polymorphism indicated that the tagged gene was both proportionally and highly expressed and that downstream processing and/or termination were critical to U3 snoRNA stability. In contrast, direct measurements of the total cellular U3 snoRNA showed essentially normal levels of mature RNA, although measurements of precursor levels confirmed a highly expressed gene construct. Taken together, the results indicated that the steady state amounts of mature U3 snoRNA were primarily regulated at the post-transcriptional level. This regulatory mechanism prevents over-accumulation of the cellular U3 snoRNA and can efficiently degrade mutant RNA molecules. Together with past studies on other 3' extended RNA precursors, the results support post-transcriptional regulation as a quality control mechanism in which appropriate amounts of functional RNA are stabilized by protein interaction while excess or defective RNA is rapidly degraded. Precursor processing in vitro and mutational analyses were consistent with this model.