Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders ...the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and κ light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics.
Traditional approaches for development of antibodies are poorly suited to combating the emergence of novel pathogens, as they require multiple steps of laborious optimization and process adaptation for clinical development. Here, we describe the simultaneous use of two state-of-the-art technologies to rapidly generate and validate antibodies against Middle East Respiratory Syndrome coronavirus (MERS-CoV), following a highly optimized process that links immunization to production of clinical material grade antibodies and developed promising clinical candidates for prophylaxis and treatment of MERS-CoV, and a humanized mouse model of infection that was used to evaluate our therapeutics. This study forms the basis for a rapid response to address the public threat resulting from emerging coronaviruses or other pathogens that pose a serious threat to human health in the future.
The Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging highly pathogenic virus causing almost 50 % lethality in infected individuals. The development of a small-animal model ...is critical for the understanding of this virus and to aid in development of countermeasures against MERS-CoV. We found that BALB/c, 129/SvEv and 129/SvEv STAT1 knockout mice are not permissive to MERS-CoV infection. The lack of infection may be due to the low level of mRNA and protein for the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4), in the lungs of mice. The low level of DPP4 in the lungs likely contributes to the lack of viral replication in these mouse models and suggests that a transgenic mouse model expressing DPP4 to higher levels is necessary to create a mouse model for MERS-CoV.
Effector T cell differentiation requires the simultaneous integration of multiple, and sometimes opposing, cytokine signals. We demonstrated mTOR's role in dictating the outcome of T cell fate. ...mTOR-deficient T cells displayed normal activation and IL-2 production upon initial stimulation. However, such cells failed to differentiate into T helper 1 (Th1), Th2, or Th17 effector cells. The inability to differentiate was associated with decreased STAT transcription factor activation and failure to upregulate lineage-specific transcription factors. Under normally activating conditions, T cells lacking mTOR differentiated into Foxp3+ regulatory T cells. This was associated with hyperactive Smad3 activation in the absence of exogenous TGF-β. Surprisingly, T cells selectively deficient in TORC1 do not divert to a regulatory T cell pathway, implicating both TORC1 and TORC2 in preventing the generation of regulatory T cells. Overall, our studies suggest that mTOR kinase signaling regulates decisions between effector and regulatory T cell lineage commitment.
The recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV), a betacoronavirus, is associated with severe pneumonia and renal failure. The environmental origin of MERS-CoV is as yet ...unknown; however, its genome sequence is closely related to those of two bat coronaviruses, named BtCoV-HKU4 and BtCoV-HKU5, which were derived from Chinese bat samples. A hallmark of highly pathogenic respiratory viruses is their ability to evade the innate immune response of the host. CoV accessory proteins, for example those from severe acute respiratory syndrome CoV (SARS-CoV), have been shown to block innate antiviral signalling pathways. MERS-CoV, similar to SARS-CoV, has been shown to inhibit type I IFN induction in a variety of cell types in vitro. We therefore hypothesized that MERS-CoV and the phylogenetically related BtCoV-HKU4 and BtCoV-HKU5 may encode proteins with similar capabilities. In this study, we have demonstrated that the ORF4b-encoded accessory protein (p4b) of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 may indeed facilitate innate immune evasion by inhibiting the type I IFN and NF-κB signalling pathways. We also analysed the subcellular localization of p4b from MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 and demonstrated that all are localized to the nucleus.
Viral proteins must intimately interact with the host cell machinery during virus replication. Here, we used the yeast
as a system to identify novel functional interactions between viral proteins and ...eukaryotic cells. Our work demonstrates that when the Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a accessory gene is expressed in yeast it causes a slow-growth phenotype. ORF4a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between ORF4a and eukaryotic cells. Using the slow-growth phenotype as a reporter of ORF4a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the YDL042C/SIR2 yeast gene as a suppressor of ORF4a function. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We found that when SIRT1 was inhibited by either chemical or genetic manipulation, there was reduced MERS-CoV replication, suggesting that SIRT1 is a proviral factor for MERS-CoV. Moreover, ORF4a inhibited SIRT1-mediated modulation of NF-κB signaling, demonstrating a functional link between ORF4a and SIRT1 in mammalian cells. Overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. We also demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in cells.
Middle East respiratory syndrome coronavirus (MERS-CoV) initially emerged in 2012 and has since been responsible for over 2,300 infections, with a case fatality ratio of approximately 35%. We have used the highly characterized model system of
to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. We identify a functional link between the MERS-CoV ORF4a proteins and the YDL042C/SIR2 yeast gene. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in mammalian cells.
Middle East respiratory syndrome coronavirus (MERS-CoV) is an important emerging pathogen that was first described in 2012. While the cell surface receptor for MERS-CoV has been identified as ...dipeptidyl peptidase 4 (DPP4), the mouse DPP4 homologue does not allow virus entry into cells. Therefore, development of mouse models of MERS-CoV has been hampered by the fact that MERS-CoV does not replicate in commonly available mouse strains. We have previously described a mouse model in which mDPP4 was replaced with hDPP4 such that hDPP4 is expressed under the endogenous mDPP4 promoter. In this study, we used this mouse model to analyze the host response to MERS-CoV infection using immunological assays and transcriptome analysis. Depletion of CD4
T cells, CD8
T cells, or macrophages has no effect on MERS-CoV replication in the lungs of infected mice. However, we found that depletion of CD8
T cells protects and depletion of macrophages exacerbates MERS-CoV-induced pathology and clinical symptoms of disease. Overall, we demonstrate an important role for the inflammatory response in regulating MERS-CoV pathogenesis in vivo IMPORTANCE: The Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus that emerged from zoonotic sources in 2012. Human infections are still occurring throughout Saudi Arabia at a 38% case fatality rate, with the potential for worldwide spread via air travel. In this work, we identify the host response to the virus and identify inflammatory pathways and cell populations that are critical for protection from severe lung disease. By understanding the immune response to MERS-CoV we can develop targeted therapies to inhibit pathogenesis in the future.
Effector T cell differentiation requires the simultaneous integration of multiple, and sometimes opposing, cytokine signals. We demonstrate that mTOR plays a role in dictating the outcome of T cell ...fate. mTOR deficient T cells display normal activation and IL-2 production upon initial stimulation. However, such cells fail to differentiate into Th1, Th2 or Th17 effector T cells under skewing conditions. The inability to differentiate is associated with a decrease in STAT activation and failure to upregulate lineage specific transcription factors. Under all normally
activating
conditions, T cells lacking mTOR differentiate into Foxp3
+
regulatory cells. This differentiation is associated with hyperactive Smad3 activation in the absence of exogenous TGF-β. Surprisingly, T cells in which TORC1 activity has been selectively deleted do not divert to a regulatory T cell pathway, revealing an unappreciated role for TORC2 signaling in preventing the generation of regulatory T cells. Overall our studies suggest that differential TORC1 and TORC2 signaling regulate decisions between effector and regulatory T cell lineage commitment.
The transcription factor GATA1 helps regulate the expression of thousands of genes involved in blood development, by binding to single or double GATA sites on DNA. An important part of gene ...activation is chromatin looping, the bringing together of DNA elements that lie up to many thousands of basepairs apart in the genome. It was recently suggested, based on studies of the closely related protein GATA3, that GATA‐mediated looping may involve interactions of each of two zinc fingers (ZF) with distantly spaced DNA elements. Here we present a structure of the GATA1 ZF region bound to pseudopalindromic double GATA site DNA, which is structurally equivalent to a recently‐solved GATA3‐DNA complex. However, extensive analysis of GATA1‐DNA binding indicates that although the N‐terminal ZF (NF) can modulate GATA1‐DNA binding, under physiological conditions the NF binds DNA so poorly that it cannot play a direct role in DNA‐looping. Rather, the ability of the NF to stabilize transcriptional complexes through protein–protein interactions, and thereby recruit looping factors such as Ldb1, provides a more compelling model for GATA‐mediated looping.
Interactive Figure 1
Objective
To determine whether there are differences in baseline psychological and behavioral characteristics between individuals with severe obesity who chose a surgical or nonsurgical intervention ...for weight loss.
Methods
The current study utilized data from a larger study funded by a state insurance company and is unique in that the insurance company funded the weight loss interventions. Participants indicated their preferred method of weight loss, and completed several self‐report psychological questionnaires, as well as demographic information.
Results
Participants (N = 605) were 58.8% Caucasian and mostly (86%) female. Logistic regression results indicated that an increased number of weight loss attempts, and select other measures of eating behavior and quality of life may influence individuals’ selection for either surgical or nonsurgical treatments for weight loss.
Conclusions
Practitioners should pay particular attention to these baseline characteristics that influence choice to examine potential characteristics that may influence the success of these weight loss treatments.
Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the ...phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a
DNA methyltransferase 3b
allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked
Hprt
gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.
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