Mouse-adapted Strain Research Articles

A Thermostable mRNA Vaccine against COVID-19

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

Incomplete glycosylation during prion infection unmasks a prion protein epitope that facilitates prion detection and strain discrimination

The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.

Relative contribution of nonstructural protein 1 in dengue pathogenesis.

Dengue is a major public health concern in the tropical and subtropical world, with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of subunit vaccine approaches, and nonstructural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non-mouse-adapted DENV2 strains that are highly virulent. Exogenous administration of purified NS1 also failed to worsen in vivo vascular leakage in sublethally infected mice. Neither method of NS1 immune neutralization changed the disease outcome of a chimeric strain expressing a vascular leak-potent NS1. Instead, virus chimerization involving the prME structural region indicated that these proteins play a critical role in driving in vivo fitness and virulence of the virus, through induction of key proinflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain dependent, which warrants reevaluation of NS1 as a universal dengue vaccine candidate.

Discordant rearrangement of primary and anamnestic CD8+ T cell responses to influenza A viral epitopes upon exposure to bacterial superantigens: Implications for prophylactic vaccination, heterosubtypic immunity and superinfections.

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vβ makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.

Why Aren't People Living with HIV at Higher Risk for Developing Severe Coronavirus Disease 2019 (COVID-19)?

Why Aren't People Living with HIV at Higher Risk for Developing Severe Coronavirus Disease 2019 (COVID-19)?

Region-Specific Response of Astrocytes to Prion Infection.

Chronic neuroinflammation involves reactive microgliosis and astrogliosis, and is regarded as a common pathological hallmark of neurodegenerative diseases including Alzheimer’s, Parkinson’s, ALS and prion diseases. Reactive astrogliosis, routinely observed immunohistochemically as an increase in glial fibrillary acidic protein (GFAP) signal, is a well-documented feature of chronic neuroinflammation associated with neurodegenerative diseases. Recent studies on single-cell transcriptional profiling of a mouse brain revealed that, under normal conditions, several distinct subtypes of astrocytes with regionally specialized distribution exist. However, it remains unclear whether astrocytic response to pro-inflammatory pathological conditions is uniform across whole brain or is region-specific. The current study compares the response of microglia and astrocytes to prions in mice infected with 22L mouse-adapted prion strain. While the intensity of reactive microgliosis correlated well with the extent of PrPSc deposition, reactive astrogliosis displayed a different, region-specific pattern. In particular, the thalamus and stratum oriens of hippocampus, which are both affected by 22L prions, displayed strikingly different response of astrocytes to PrPSc. Astrocytes in stratum oriens of hippocampus responded to accumulation of PrPSc with visible hypertrophy and increased GFAP, while in the thalamus, despite stronger PrPSc signal, the increase of GFAP was milder than in hippocampus, and the change in astrocyte morphology was less pronounced. The current study suggests that astrocyte response to prion infection is heterogeneous and, in part, defined by brain region. Moreover, the current work emphasizes the needs for elucidating region-specific changes in functional states of astrocytes and exploring the impact of these changes to chronic neurodegeneration.

Discrimination of Prion Strain Targeting in the Central Nervous System via Reactive Astrocyte Heterogeneity in CD44 Expression.

Prion diseases or transmissible spongiform encephalopathies are fatal, progressive, neurodegenerative, protein-misfolding disorders. Prion diseases may arise spontaneously, be inherited genetically or be acquired by infection and affect a variety of mammalian species including humans. Prion infections in the central nervous system (CNS) cause extensive neuropathology, including abnormal accumulations of misfolded host prion protein, vacuolar change resulting in sponge-like (spongiform) appearance of CNS tissue, neurodegeneration and reactive glial responses. Many different prion agent strains exist and these can differ based on disease duration, clinical signs and the targeting and distribution of the neuropathology in distinct brain areas. Reactive astrocytes are a prominent feature in the prion disease affected CNS as revealed by distinct morphological changes and upregulation of glial fibrillary acidic protein (GFAP). The CD44 antigen is a transmembrane glycoprotein involved in cell-cell interactions, cell adhesion and migration. Here we show that CD44 is also highly expressed in a subset of reactive astrocytes in regions of the CNS targeted by prions. Astrocyte heterogeneity revealed by differential CD44 upregulation occurs coincident with the earliest neuropathological changes during the pre-clinical phase of disease, and is not affected by the route of infection. The expression and distribution of CD44 was compared in brains from a large collection of 15 distinct prion agent strains transmitted to mice of different prion protein (Prnp) genotype backgrounds. Our data show that the pattern of CD44 upregulation observed in the hippocampus in each prion agent strain and host Prnp genotype combination was unique. Many mouse-adapted prion strains and hosts have previously been characterized based on the pattern of the distribution of the spongiform pathology or the misfolded PrP deposition within the brain. Our data show that CD44 expression also provides a reliable discriminatory marker of prion infection with a greater dynamic range than misfolded prion protein deposition, aiding strain identification. Together, our data reveal CD44 as a novel marker to detect reactive astrocyte heterogeneity during CNS prion disease and for enhanced identification of distinct prion agent strains.

PA-X antagonises MAVS-dependent accumulation of early type I interferon messenger RNAs during influenza A virus infection

The sensing of viral nucleic acids by the innate immune system activates a potent antiviral response in the infected cell, a key component of which is the expression of genes encoding type I interferons (IFNs). Many viruses counteract this response by blocking the activation of host nucleic acid sensors. The evolutionarily conserved influenza A virus (IAV) protein PA-X has been implicated in suppressing the host response to infection, including the expression of type I IFNs. Here, we characterise this further using a PA-X-deficient virus of the mouse-adapted PR8 strain to study activation of the innate immune response in a mouse model of the early response to viral infection. We show that levels of Ifna4 and Ifnb1 mRNAs in the lungs of infected mice were elevated in the absence of PA-X and that this was completely dependent on MAVS. This therefore suggests a role for PA-X in preventing the accumulation of early type I IFN mRNAs in the lung during IAV infection.

The cell type resolved mouse transcriptome in neuron-enriched brain tissues from the hippocampus and cerebellum during prion disease

Multiple cell types and complex connection networks are an intrinsic feature of brain tissue. In this study we used expression profiling of specific microscopic regions of heterogeneous tissue sections isolated by laser capture microdissection (LCM) to determine insights into the molecular basis of brain pathology in prion disease. Temporal profiles in two mouse models of prion disease, bovine spongiform encephalopathy (BSE) and a mouse-adapted strain of scrapie (RML) were performed in microdissected regions of the CA1 hippocampus and granular layer of the cerebellum which are both enriched in neuronal cell bodies. We noted that during clinical disease the number of activated microglia and astrocytes that occur in these areas are increased, thereby likely diluting the neuronal gene expression signature. We performed a comparative analysis with gene expression profiles determined from isolated populations of neurons, microglia and astrocytes to identify transcripts that are enriched in each of these cell types. Although the incubation periods of these two models are quite different, over 300 days for BSE and ~160 days for RML scrapie, these regional microdissections revealed broadly similar profiles. Microglial and astrocyte-enriched genes contributed a profound inflammatory profile consisting of inflammatory cytokines, genes related to phagocytosis, proteolysis and genes coding for extracellular matrix proteins. CA1 pyramidal neurons displayed a net upregulation of transcription factors and stress induced genes at pre-clinical stages of disease while all tissues showed profound decrease of overlapping genes related to neuronal function, in particular transcripts related to neuronal communication including glutamate receptors, phosphatase subunits and numerous synapse-related markers. Of note, we found a small number of genes expressed in neurons that were upregulated during clinical disease including, COX6A2, FZD9, RXRG and SOX11, that may be biomarkers of neurodegeneration.

The XRE Family Transcriptional Regulator SrtR in Streptococcus suis Is Involved in Oxidant Tolerance and Virulence

Streptococcus suis is a zoonotic pathogen that harbors anti-oxidative stress genes, which have been reported to be associated with virulence. Serial passage has been widely used to obtain phenotypic variant strains to investigate the functions of important genes. In the present study, S. suis serotype 9 strain DN13 was serially passaged in mice 30 times. The virulence of a single colony from passage 10 (SS9-P10) was found to increase by at least 140-fold as indicated by LD50 values, and the increased virulence was stable for single colonies from passage 20 (SS0-P20) and 30 (SS0-P30). Compared to the parental strain, the mouse-adapted strains were more tolerant to oxidative and high temperature stress. Genome-wide analysis of nucleotide variations found that reverse mutations occurred in seven genes, as indicated by BLAST analysis. Three of the reverse mutation genes or their homologs in other bacteria were reported to be virulence-associated, including ideSsuis in S. suis, a homolog of malR of Streptococcus pneumoniae, and a homolog of the prepilin peptidase-encoding gene in Legionella pneumophila. However, these genes were not involved in the stress response. Another gene, srtR (stress response transcriptional regulator), encoding an XRE family transcriptional regulator, which had an internal stop in the parental strain, was functionally restored in the adapted strains. Further analysis of DN13 and SS9-P10-background srtR-knock-out and complementing strains supported the contribution of this gene to stress tolerance in vitro and virulence in mice. srtR and its homologs are widely distributed in Gram-positive bacteria including several important human pathogens such as Enterococcus faecium and Clostridioides difficile, indicating similar functions in these bacteria. Taken together, our study identified the first member of the XRE family of transcriptional regulators that is involved in stress tolerance and virulence. It also provides insight into the mechanism of enhanced virulence after serial passage in experimental animals.

Re-transmissibility of mouse-adapted ME7 scrapie strain to ovine PrP transgenic mice.

Scrapie is a mammalian transmissible spongiform encephalopathy or prion disease that predominantly affects sheep and goats. Scrapie has been shown to overcome the species barrier via experimental infection of other rodents. To confirm the re-transmissibility of the mouse-adapted ME7 scrapie strain to ovine prion protein (PrP) transgenic mice, mice of an ovinized transgenic mouse line carrying the Suffolk sheep PrP gene that contained the A136 R154 Q171/ARQ allele were intracerebrally inoculated with brain homogenates obtained from terminally ill ME7-infected C57BL/6J mice. Herein, we report that the mouse-adapted ME7 scrapie strain was successfully re-transmitted to the transgenic mice expressing ovine PrP. In addition, we observed changes in the incubation period, glycoform profile, and pattern of scrapie PrP (PrPSc) deposition in the affected brains. PrPSc deposition in the hippocampal region of the brain of 2nd-passaged ovine PrP transgenic mice was accompanied by plaque formation. These results reveal that the mouse-adapted ME7 scrapie strain has the capacity to act as a template for the conversion of ovine normal monomeric precursors into a pathogenic form in ovine PrP transgenic mice. The change in glycoform pattern and the deposition of plaques in the hippocampal region of the brain of the 2nd-passaged PrP transgenic mice are most likely cellular PrP species dependent rather than being ME7 scrapie strain encoded.

Antiviral effects against influenza A virus infection by a short hairpin RNA targeting the non-coding terminal region of the viral nucleoprotein gene.

RNA interference (RNAi) can inhibit Influenza A virus (IAV) infection in a gene-specific manner. In this study, we constructed a transgene expressing a short hairpin RNA (shRNA) that targets the noncoding region of the IAV RNA gene encoding nucleoprotein (NP). To investigate the antiviral effects of the shRNA, we generated two transgenic mouse lines with this transgene. Unfortunately, there was no apparent difference in IAV resistance between transgenic and non-transgenic littermates. To further investigate the antiviral effects of the shRNA, we prepared mouse embryonic fibroblasts (MEFs) from transgenic and non-transgenic mice. In experimental infections using these MEFs, virus production of mouse-adapted IAV strain A/Puerto Rico/8/1934 (PR8) in the transgenic MEFs was suppressed by means of the down-regulation of the viral RNA gene transcription in the early stages of infection in comparison with non-transgenic MEFs. These results indicated that expression of the shRNA was able to confer antiviral properties against IAVs to MEFs, although the effects were limited. Our findings suggest that the shRNA targeting the noncoding region of the viral RNA (vRNA) of NP might be a supporting tool in developing influenza-resistant poultry.

The African strain of Zika virus causes more severe in utero infection than Asian strain in a porcine fetal transmission model

ABSTRACT Studies in mice showed that African Zika virus (ZIKV) strains cause more damage in embryos. These studies, however, were limited to the mouse-adapted African MR766 strain or infection at early gestation. Here, we compared infection of Asian and African strains in the fetal pig model at midgestation. Both strains caused fetal infection. ZIKV was detected in placenta, amniotic membrane, amniotic fluid, fetal blood, and brain. The African strain produced more vigorous in utero infection as represented by more efficient virus transmission between siblings, and higher viral loads in fetal organs and membranes. Infection with both strains was associated with reduced fetal brain weight and increased number of placental CD163-positive cells, as well as elevated in utero interferon alpha and cortisol levels. This is the first large animal model study which demonstrated that African strain of ZIKV, with no passage history in experimental animals, can cause persistent infection in fetuses and fetal membranes at midgestation. Our studies also suggest that similar to Asian strains, ZIKV of African lineage might cause silent pathology which is difficult to identify in deceptively healthy fetuses. The findings emphasize the need for further studies to highlight the impact of ZIKV heterogeneity on infection outcomes during pregnancy.

Immunocompetent and Immunodeficient Mouse Models for Enterovirus 71 Pathogenesis and Therapy.

Enterovirus 71 (EV71) is a global health threat. Children infected with EV71 could develop hand-foot-and-mouth disease (HFMD), encephalitis, paralysis, pulmonary edema, and death. At present, no effective treatment for EV71 is available. We reviewed here various mouse models for EV71 pathogenesis and therapy. Earlier studies relied on the use of mouse-adapted EV71 strains. To avoid artificial mutations arising de novo during the serial passages, recent studies used EV71 clinical isolates without adaptation. Several human receptors for EV71 were shown to facilitate viral entry in cell culture. However, in vivo infection with human SCARB2 receptor transgenic mice appeared to be more limited to certain strains and genotypes of EV71. Efficacy of oral infection in these transgenic models is extremely low. Intriguingly, despite the lack of human receptors, immunodeficient neonatal mouse models can still be infected with EV71 clinical isolates via oral or intraperitoneal routes. Crossbreeding between SCARB2 transgenic and stat1 knockout mice generated a more sensitive and user-friendly hybrid mouse model. Infected hybrid mice developed a higher incidence and earlier onset of CNS disease and death. Different pathogenesis profiles were observed in models deficient in various arms of innate or humoral immunity. These models are being actively used for antiviral research.

Tropism for Spasmolytic Polypeptide-Expressing Metaplasia Allows Helicobacter pylori to Expand Its Intragastric Niche

In patients with chronic Helicobacter pylori (H pylori) infection, parietal and chief cell atrophy in the gastric corpus, a process known as spasmolytic polypeptide-expressing metaplasia (SPEM), increases the risk for progression to cancer. The relation between H pylori and these metaplastic changes is unclear. We investigated whether Hpylori localizes to regions of SPEM. We developed an in situ adherence assay in which we incubated H pylori with free-floating tissue sections from the gastric corpora of mice; we assessed H pylori distribution along the gastric unit by immunofluorescence. We analyzed the interactions of H pylori with tissue collected from mice with acute SPEM, induced by high-dose tamoxifen. We also evaluated how adhesin-deficient H pylori strains, chemical competition assays, and epithelial glycosylation affected H pylori adhesion to SPEM glands. Mice colonized with the mouse-adapted PMSS1 strain were analyzed for H pylori colonization in vivo during tamoxifen-induced SPEM or after decrease of stomach acid with omeprazole. Compared with uninjured glands, H pylori penetrated deep within SPEM glands, in situ, through interaction of its adhesin, SabA, with sialyl-Lewis X, which expanded in SPEM. H pylori markedly increased gastric corpus colonization when SPEM was induced, but this proximal spread reversed in mice allowed to recover from SPEM. Decreasing corpus acidity also promoted proximal spread. However, Hpylori penetrated deep within corpus glands invivo only when sialyl-Lewis X expanded during SPEM. Helicobacter pylori differentially binds SPEM glands in situ and in mice, in large part by interacting with sialyl-Lewis X. Our findings indicate that H pylori expands its niche into the gastric corpus by promoting and exploiting epithelial metaplastic changes that can lead to tumorigenesis.

Lipopolysaccharide Structural Differences between Western and Asian Helicobacter pylori Strains.

Recent structural analysis of the lipopolysaccharide (LPS) isolated from Helicobacter pylori G27 wild-type and O-antigen ligase mutant resulted in the redefinition of the core-oligosaccharide and O-antigen domains. The short core-oligosaccharide (Glc–Gal–Hep-III–Hep-II–Hep-I–KDO) and its attached trisaccharide (Trio, GlcNAc–Fuc–Hep) appear to be highly conserved structures among H. pylori strains. The G27 LPS contains a linear glucan–heptan linker between the core-Trio and distal Lewis antigens. This linker domain was commonly identified in Western strains. In contrast, out of 12 partial LPS structures of Asian strains, none displayed the heptan moiety, despite the presence of Lewis antigens. This raises the question of how Lewis antigens are attached to the Trio, and whether the LPS structure of Asian strains contain another linker. Of note, a riban was identified as a linker in LPS of the mouse-adapted SS1 strain, suggesting that alternative linker structures can occur. In summary, additional full structural analyses of LPS in Asian strains are required to assess the presence or absence of an alternative linker in these strains. It will also be interesting to study the glucan-heptan linker moieties in pathogenesis as H. pylori infections in Asia are usually more symptomatic than the ones presented in the Western world.

Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species.

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.

Inflammatory response of microglia to prions is controlled by sialylation of PrPSc

Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer’s, Parkinson’s or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκβα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

In vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs

The pathophysiological changes that occur in lungs infected with influenza viruses are poorly understood. Here we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity. This approach allowed us to monitor and correlate several parameters and physiological changes including the spread of infection, pulmonary permeability, pulmonary perfusion speed, number of recruited neutrophils in infected lungs, and neutrophil motion in the lungs of live mice. Several physiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N1 influenza virus compared with those infected with a mouse-adapted human strain. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infections.