Host cell-intrinsic innate immune recognition of SARS-CoV-2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged at the end of 2019 and caused the pandemic of coronavirus disease 2019 (COVID-19). Basic and clinical investigations indicate that severe forms of COVID-19 are due in part to dysregulated immune responses to virus infection. The innate immune system is the first line of host defense against most virus infections, with pathogen recognition receptors detecting SARS-CoV-2 RNA and protein components and initiating pro-inflammatory and antiviral responses. Notwithstanding this response, SARS-CoV-2 proteins evade, inhibit, and skew innate immune signaling early in infection. In this review, we highlight the components of cell-based recognition of SARS-CoV-2 infection and the mechanisms employed by the virus to modulate these innate immune host defense pathways

USING SHAPE-MaP TO IDENTIFY FUNCTIONAL RNA SECONDARY STRUCTURES IN RNA VIRUSES

Alphaviruses are a genus of arboviruses often transmitted by mosquitos. Alphaviruses are responsible for multiple outbreaks over the last two decades and continue to pose a serious threat to human health. The 2013 outbreak of chikungunya virus (CHIKV), the most notable alphavirus, caused over one million infections. Despite the frequency with which these viruses re-emerge, there are no effective therapies or vaccines against alphaviruses. Like other RNA viruses, alphavirus genomes contain functionally important RNA secondary structures that contribute to immune evasion, RNA transcription, RNA translation, and virion assembly. However, very little of alphavirus genomes have been characterized due to a previous inability to accurately and quickly model long RNAs. This work used the RNA structure probing technique SHAPE-MaP to produce experimentally informed RNA secondary structure models of multiple RNA virus genomes. We probed genomes of closely related alphaviruses to identify conserved structured and unstructured regions. We found that alphaviruses are structurally unique and most conserved structured regions fold into distinct RNA secondary structures. After we identified a novel functionally important RNA secondary structure specific to Sindbis virus, we revised our approach to identify regions within each virus likely to fold into a specific conformation. We identified 23 regions of the CHIKV genome that were specifically structured. The four previously known RNA secondary structures were included in the 23 regions identified, validating the approach. Further, we demonstrated that one of the uncharacterized structured regions enhanced virus replication. Lastly, we demonstrated our approach to structure identification and testing was applicable to RNA viruses beyond the alphavirus genus using Zika virus, a flavivirus responsible for a large outbreak in 2015. For each of our studies we used silent structure disrupting mutations to assess RNA structure without affecting protein coding sequence, so structures could be assessed in the context of infection. These findings improve our understanding of known pathogenic RNA viruses and provide an approach to quickly study and assess future emerging RNA viruses. A more comprehensive knowledge of functionally important RNA structures in viruses could be used to design safer live attenuated vaccines or develop new RNA-binding small molecule therapies.Doctor of Philosoph

Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees

Women's preferences for a new contraceptive under development: an exploratory study

ObjectiveCurrently available contraceptive methods do not meet the needs of all users. We sought to explore preferences of potential end-users regarding an on-demand, non-hormonal female contraceptive currently under development, using a web-based survey.Study designWe recruited respondents for an exploratory survey via web link on Amazon Mechanical Turk (MTurk). Individuals were eligible if they were 18–44 years of age, identified as cis-gender female, were English-speaking, not pregnant, and had used barrier contraception previously. Respondents provided demographic characteristics and a basic reproductive history. We then provided a brief description of the potential contraceptive. Respondents were asked about their interest in the proposed contraceptive and preferences for method attributes.ResultsA total of 500 respondents completed the survey. Three-quarters of respondents were <35 years of age and 48.2% were currently using a barrier contraceptive method. Three-fourths of respondents (73.8%) expressed interest in using the contraceptive under development. The majority wanted the method to be small (≤2 inches), rod-shaped, and low cost (<$5 per use). More than half (59.4%) said it was important to be able to use the method without partners’ knowledge. The most reported potential concerns were vaginal irritation (51.6%) and lack of effectiveness (46.4%). Sixty percent of respondents were confident they could use the method correctly.DiscussionAvailable contraceptive methods lack attributes preferred by some users. Development of new contraceptives frequently does not involve end-user input early in the development process. Individuals in this sample displayed interest in the proposed contraceptive and expressed preferences that can inform the further development of this method

Structural divergence creates new functional features in alphavirus genomes

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins

Nanoformulated Remdesivir with Extremely Low Content of Poly(2-oxazoline)-Based Stabilizer for Aerosol Treatment of COVID-19

The rise of the novel virus SARS-CoV2 which causes the disease known as COVID-19 has led to a global pandemic claiming millions of lives. With no clinically approved treatment for COVID-19, physicians initially struggled to treat the disease, and a need remains for improved antiviral therapies in this area. It is conceived early in the pandemic that an inhalable formulation of the drug remdesivir which directly targets the virus at the site of infection could improve therapeutic outcomes in COVID-19. A set of requirements are developed that would be conducive to rapid drug approval: 1) try to use GRAS reagents 2) minimize excipient concentration and 3) achieve a working concentration of 5 mg/mL remdesivir to obtain a deliverable dose which is 5-10% of the IV dose. In this work, it is discovered that Poly(2-oxazoline) block copolymers can stabilize drug nanocrystal suspensions and provide suitable formulation characteristics for aerosol delivery while maintaining antiviral efficacy. The authors believe POx block copolymers can be used as a semi-ubiquitous stabilizer for the rapid development of nanocrystal formulations for new and existing diseases

The use of economic evaluation in CAM: an introductory framework

Background For CAM to feature prominently in health care decision-making there is a need to expand the evidence-base and to further incorporate economic evaluation into research priorities. In a world of scarce health care resources and an emphasis on efficiency and clinical efficacy, CAM, as indeed do all other treatments, requires rigorous evaluation to be considered in budget decision-making. Methods Economic evaluation provides the tools to measure the costs and health consequences of CAM interventions and thereby inform decision making. This article offers CAM researchers an introductory framework for understanding, undertaking and disseminating economic evaluation. The types of economic evaluation available for the study of CAM are discussed, and decision modelling is introduced as a method for economic evaluation with much potential for use in CAM. Two types of decision models are introduced, decision trees and Markov models, along with a worked example of how each method is used to examine costs and health consequences. This is followed by a discussion of how this information is used by decision makers. Conclusions Undoubtedly, economic evaluation methods form an important part of health care decision making. Without formal training it can seem a daunting task to consider economic evaluation, however, multidisciplinary teams provide an opportunity for health economists, CAM practitioners and other interested researchers, to work together to further develop the economic evaluation of CAM

Forward genetic screen of homeostatic antibody levels in the Collaborative Cross identifies MBD1 as a novel regulator of B cell homeostasis

Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL map to genome regions of known immunological significance (major histocompatibility and immunoglobulin heavy chain locus), Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and identify Methyl-CpG binding domain protein 1 under this locus as a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells

Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like β-Coronaviruses

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human, bat, and murine β-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in β-coronavirus replication. Spike protein endocytosis was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for the development of anti-SARS-like β-coronavirus drugs

Host Genetic Variation Impacts SARS-CoV-2 Vaccination Response in the Diversity Outbred Mouse Population

The COVID-19 pandemic led to the rapid and worldwide development of highly effective vaccines against SARS-CoV-2. However, there is significant individual-to-individual variation in vaccine efficacy due to factors including viral variants, host age, immune status, environmental and host genetic factors. Understanding those determinants driving this variation may inform the development of more broadly protective vaccine strategies. While host genetic factors are known to impact vaccine efficacy for respiratory pathogens such as influenza and tuberculosis, the impact of host genetic variation on vaccine efficacy against COVID-19 is not well understood. To model the impact of host genetic variation on SARS-CoV-2 vaccine efficacy, while controlling for the impact of non-genetic factors, we used the Diversity Outbred (DO) mouse model. We found that DO mice immunized against SARS-CoV-2 exhibited high levels of variation in vaccine-induced neutralizing antibody responses. While the majority of the vaccinated mice were protected from virus-induced disease, similar to human populations, we observed vaccine breakthrough in a subset of mice. Importantly, we found that this variation in neutralizing antibody, virus-induced disease, and viral titer is heritable, indicating that the DO serves as a useful model system for studying the contribution of genetic variation of both vaccines and disease outcomes