Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Fungal-mediated lung allergic airway disease: The critical role of macrophages and dendritic cells.

Fungi are abundant in the environment, causing our lungs to be constantly exposed to a diverse range of species. While the majority of these are cleared effectively in healthy individuals, constant exposure to spores (especially Aspergillus spp.) can lead to the development of allergic inflammation that underpins and worsen diseases such as asthma. Despite this, the precise mechanisms that underpin the development of fungal allergic disease are poorly understood. Innate immune cells, such as macrophages (MΦs) and dendritic cells (DCs), have been shown to be critical for mediating allergic inflammation to a range of different allergens. This review will focus on the crucial role of MΦ and DCs in mediating antifungal immunity, evaluating how these immune cells mediate allergic inflammation within the context of the lung environment. Ultimately, we aim to highlight important future research questions that will lead to novel therapeutic strategies for fungal allergic diseases.

Dual-locus, dual-HDR editing permits efficient generation of antigen-specific regulatory T cells with robust suppressive activity.

Adoptive regulatory T (Treg) cell therapy is predicted to modulate immune tolerance in autoimmune diseases, including type 1 diabetes (T1D). However, the requirement for antigen (ag) specificity to optimally orchestrate tissue-specific, Treg cell-mediated tolerance limits effective clinical application. To address this challenge, we present a single-step, combinatorial gene editing strategy utilizing dual-locus, dual-homology-directed repair (HDR) to generate and specifically expand ag-specific engineered Treg (EngTreg) cells derived from donor CD4+ T cells. Concurrent delivery of CRISPR nucleases and recombinant (r)AAV homology donor templates targeting FOXP3 and TRAC was used to achieve three parallel goals: enforced, stable expression of FOXP3; replacement of the endogenous T cell receptor (TCR) with an islet-specific TCR; and selective enrichment of dual-edited cells. Each HDR donor template contained an alternative component of a heterodimeric chemically inducible signaling complex (CISC), designed to activate interleukin-2 (IL-2) signaling in response to rapamycin, promoting expansion of only dual-edited EngTreg cells. Using this approach, we generated purified, islet-specific EngTreg cells that mediated robust direct and bystander suppression of effector T (Teff) cells recognizing the same or a different islet antigen peptide, respectively. This platform is broadly adaptable for use with alternative TCRs or other targeting moieties for application in tissue-specific autoimmune or inflammatory diseases.

A chemically inducible IL-2 receptor signaling complex allows for effective in vitro and in vivo selection of engineered CD4+ T cells.

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.

Pathogenesis of Respiratory Viral and Fungal Coinfections.

Individuals suffering from severe viral respiratory tract infections have recently emerged as "at risk" groups for developing invasive fungal infections. Influenza virus is one of the most common causes of acute lower respiratory tract infections worldwide. Fungal infections complicating influenza pneumonia are associated with increased disease severity and mortality, with invasive pulmonary aspergillosis being the most common manifestation. Strikingly, similar observations have been made during the current coronavirus disease 2019 (COVID-19) pandemic. The copathogenesis of respiratory viral and fungal coinfections is complex and involves a dynamic interplay between the host immune defenses and the virulence of the microbes involved that often results in failure to return to homeostasis. In this review, we discuss the main mechanisms underlying susceptibility to invasive fungal disease following respiratory viral infections. A comprehensive understanding of these interactions will aid the development of therapeutic modalities against newly identified targets to prevent and treat these emerging coinfections.

Simulating carbon accumulation and loss in the central Congo peatlands.

Peatlands of the central Congo Basin have accumulated carbon over millennia. They currently store some 29 billion tonnes of carbon in peat. However, our understanding of the controls on peat carbon accumulation and loss and the vulnerability of this stored carbon to climate change is in its infancy. Here we present a new model of tropical peatland development, DigiBog_Congo, that we use to simulate peat carbon accumulation and loss in a rain-fed interfluvial peatland that began forming ~20,000 calendar years Before Present (cal. yr BP, where 'present' is 1950 CE). Overall, the simulated age-depth curve is in good agreement with palaeoenvironmental reconstructions derived from a peat core at the same location as our model simulation. We find two key controls on long-term peat accumulation: water at the peat surface (surface wetness) and the very slow anoxic decay of recalcitrant material. Our main simulation shows that between the Late Glacial and early Holocene there were several multidecadal periods where net peat and carbon gain alternated with net loss. Later, a climatic dry phase beginning ~5200 cal. yr BP caused the peatland to become a long-term carbon source from ~3975 to 900 cal. yr BP. Peat as old as ~7000 cal. yr BP was decomposed before the peatland's surface became wetter again, suggesting that changes in rainfall alone were sufficient to cause a catastrophic loss of peat carbon lasting thousands of years. During this time, 6.4 m of the column of peat was lost, resulting in 57% of the simulated carbon stock being released. Our study provides an approach to understanding the future impact of climate change and potential land-use change on this vulnerable store of carbon.

Genomic Surveillance for SARS-CoV-2 Variants: Circulation of Omicron Lineages - United States, January 2022-May 2023.

CDC has used national genomic surveillance since December 2020 to monitor SARS-CoV-2 variants that have emerged throughout the COVID-19 pandemic, including the Omicron variant. This report summarizes U.S. trends in variant proportions from national genomic surveillance during January 2022-May 2023. During this period, the Omicron variant remained predominant, with various descendant lineages reaching national predominance (>50% prevalence). During the first half of 2022, BA.1.1 reached predominance by the week ending January 8, 2022, followed by BA.2 (March 26), BA.2.12.1 (May 14), and BA.5 (July 2); the predominance of each variant coincided with surges in COVID-19 cases. The latter half of 2022 was characterized by the circulation of sublineages of BA.2, BA.4, and BA.5 (e.g., BQ.1 and BQ.1.1), some of which independently acquired similar spike protein substitutions associated with immune evasion. By the end of January 2023, XBB.1.5 became predominant. As of May 13, 2023, the most common circulating lineages were XBB.1.5 (61.5%), XBB.1.9.1 (10.0%), and XBB.1.16 (9.4%); XBB.1.16 and XBB.1.16.1 (2.4%), containing the K478R substitution, and XBB.2.3 (3.2%), containing the P521S substitution, had the fastest doubling times at that point. Analytic methods for estimating variant proportions have been updated as the availability of sequencing specimens has declined. The continued evolution of Omicron lineages highlights the importance of genomic surveillance to monitor emerging variants and help guide vaccine development and use of therapeutics.

Informing Building Strategies to Reduce Infectious Aerosol Transmission Risk by Integrating DNA Aerosol Tracers with Quantitative Microbial Risk Assessment.

Using aerosol-based tracers to estimate risk of infectious aerosol transmission aids in the design of buildings with adequate protection against aerosol transmissible pathogens, such as SARS-CoV-2 and influenza. We propose a method for scaling a SARS-CoV-2 bulk aerosol quantitative microbial risk assessment (QMRA) model for impulse emissions, coughing or sneezing, with aerosolized synthetic DNA tracer concentration measurements. With point-of-emission ratios describing relationships between tracer and respiratory aerosol emission characteristics (i.e., volume and RNA or DNA concentrations) and accounting for aerosolized pathogen loss of infectivity over time, we scale the inhaled pathogen dose and risk of infection with time-integrated tracer concentrations measured with a filter sampler. This tracer-scaled QMRA model is evaluated through scenario testing, comparing the impact of ventilation, occupancy, masking, and layering interventions on infection risk. We apply the tracer-scaled QMRA model to measurement data from an ambulatory care room to estimate the risk reduction resulting from HEPA air cleaner operation. Using DNA tracer measurements to scale a bulk aerosol QMRA model is a relatively simple method of estimating risk in buildings and can be applied to understand the impact of risk mitigation efforts.

Radical rubrics: implementing the critical and creative thinking general capability through an ecological approach.

This article details how and why we have developed a flexible and responsive process-based rubric exemplar for teaching, learning, and assessing critical and creative thinking. We hope to contribute to global discussions of and efforts toward instrumentalising the challenge of assessing, but not standardising, creativity in compulsory education. Here, we respond to the key ideas of the four interrelated elements in the critical and creative thinking general capability in the Australian Curriculum learning continuum: inquiring; generating ideas, possibilities, actions; reflecting on thinking processes; and analysing, synthesising and evaluating reasoning and procedures. The rubrics, radical because they privilege process over outcome, have been designed to be used alongside the current NAPLAN tests in Years 5, 7 and 9 to build an Australian-based national creativity measure. We do so to argue the need for local and global measures of creativity in education as the first round of testing and results of the PISA Assessment of Creative Thinking approach and to contribute to the recognition of creative thinking (and doing) as a core twenty-first century literacy alongside literacy and numeracy.

Severe Acute Respiratory Syndrome Coronavirus 2 Transmission in a Georgia School District-United States, December 2020-January 2021.

BACKGROUND: To inform prevention strategies, we assessed the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and settings in which transmission occurred in a Georgia public school district. METHODS: During 1 December 2020-22 January 2021, SARS-CoV-2-infected index cases and their close contacts in schools were identified by school and public health officials. For in-school contacts, we assessed symptoms and offered SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) testing; performed epidemiologic investigations and whole-genome sequencing to identify in-school transmission; and calculated secondary attack rate (SAR) by school setting (eg, sports, elementary school classroom), index case role (ie, staff, student), and index case symptomatic status. RESULTS: We identified 86 index cases and 1119 contacts, 688 (61.5%) of whom received testing. Fifty-nine of 679 (8.7%) contacts tested positive; 15 of 86 (17.4%) index cases resulted in ≥2 positive contacts. Among 55 persons testing positive with available symptom data, 31 (56.4%) were asymptomatic. Highest SARs were in indoor, high-contact sports settings (23.8% [95% confidence interval {CI}, 12.7%-33.3%]), staff meetings/lunches (18.2% [95% CI, 4.5%-31.8%]), and elementary school classrooms (9.5% [95% CI, 6.5%-12.5%]). The SAR was higher for staff (13.1% [95% CI, 9.0%-17.2%]) vs student index cases (5.8% [95% CI, 3.6%-8.0%]) and for symptomatic (10.9% [95% CI, 8.1%-13.9%]) vs asymptomatic index cases (3.0% [95% CI, 1.0%-5.5%]). CONCLUSIONS: Indoor sports may pose a risk to the safe operation of in-person learning. Preventing infection in staff members, through measures that include coronavirus disease 2019 vaccination, is critical to reducing in-school transmission. Because many positive contacts were asymptomatic, contact tracing should be paired with testing, regardless of symptoms.

SARS-CoV-2 Delta-Omicron Recombinant Viruses, United States.

To detect new and changing SARS-CoV-2 variants, we investigated candidate Delta-Omicron recombinant genomes from Centers for Disease Control and Prevention national genomic surveillance. Laboratory and bioinformatic investigations identified and validated 9 genetically related SARS-CoV-2 viruses with a hybrid Delta-Omicron spike protein.

Genomic Surveillance for SARS-CoV-2 Variants: Predominance of the Delta (B.1.617.2) and Omicron (B.1.1.529) Variants - United States, June 2021-January 2022.

Genomic surveillance is a critical tool for tracking emerging variants of SARS-CoV-2 (the virus that causes COVID-19), which can exhibit characteristics that potentially affect public health and clinical interventions, including increased transmissibility, illness severity, and capacity for immune escape. During June 2021-January 2022, CDC expanded genomic surveillance data sources to incorporate sequence data from public repositories to produce weighted estimates of variant proportions at the jurisdiction level and refined analytic methods to enhance the timeliness and accuracy of national and regional variant proportion estimates. These changes also allowed for more comprehensive variant proportion estimation at the jurisdictional level (i.e., U.S. state, district, territory, and freely associated state). The data in this report are a summary of findings of recent proportions of circulating variants that are updated weekly on CDC's COVID Data Tracker website to enable timely public health action.† The SARS-CoV-2 Delta (B.1.617.2 and AY sublineages) variant rose from 1% to >50% of viral lineages circulating nationally during 8 weeks, from May 1-June 26, 2021. Delta-associated infections remained predominant until being rapidly overtaken by infections associated with the Omicron (B.1.1.529 and BA sublineages) variant in December 2021, when Omicron increased from 1% to >50% of circulating viral lineages during a 2-week period. As of the week ending January 22, 2022, Omicron was estimated to account for 99.2% (95% CI = 99.0%-99.5%) of SARS-CoV-2 infections nationwide, and Delta for 0.7% (95% CI = 0.5%-1.0%). The dynamic landscape of SARS-CoV-2 variants in 2021, including Delta- and Omicron-driven resurgences of SARS-CoV-2 transmission across the United States, underscores the importance of robust genomic surveillance efforts to inform public health planning and practice.

Volumetric and connectivity assessment of the caudate nucleus in California sea lions and coyotes.

In addition to a large (chimpanzee-sized) and heavily convoluted brain, one of the most striking neurobiological features in pinnipeds is the large size of the head of the caudate nucleus, which dwarfs the rest of the striatum. Although previous research has suggested carnivore striatum is small in comparison to that of primates, there are limited volumetric data on separate striatal structures in carnivores. Therefore, the apparent functional implication of a potentially hypertrophic caudate to carnivores has not been discussed. Here, for the first time, we obtained separate volumetric measurements of caudate and putamen in California sea lions and coyotes. Exemplars of both species had very large caudate nuclei, approximately 1/75th of total brain volume. In both the sea lion and coyote, the caudate dwarfed the putamen at a ratio of 13 to 1 or greater, a finding in strong contrast to measurements showing larger putamen than caudate in primates. In addition, using post-mortem diffusion tensor brain imaging, we mapped and compared white matter connections between the dorsal caudate and the motor, premotor and frontopolar, and orbitofrontal cortices in healthy adult sea lions and healthy adult coyotes. The sea lions showed some evidence of greater premotor and frontopolar connectivity. These findings bear on previously underexplored striatal characteristics of large carnivores, and we discuss potential interpretations related to cognitive flexibility and sensorimotor transformation.

Type I interferon is required for T helper (Th) 2 induction by dendritic cells.

Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN-I) in enabling this process. An IFN-I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN-I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1-/- mice were incapable of initiating Th2 responses in vivo These data demonstrate for the first time that the influence of IFN-I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.

B cells on the brain: meningeal IgA and a novel gut-brain firewall.

Fitzpatrick et al. describe how IgA secretion by B cells and plasma cells in the mengines is crucial for protection against microbial invasion into the brain and the CNS.

Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models.

Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.