SARS-CoV-2 breakthrough infection induces rapid memory and de novo T cell responses.

Although the protective role of neutralizing antibodies against COVID-19 is well established, questions remain about the relative importance of cellular immunity. Using 6 pMHC multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of CheA.

Motile bacteria have a chemotaxis system that enables them to sense their environment and direct their swimming toward favorable conditions. Chemotaxis involves a signaling process in which ligand binding to the extracellular domain of the chemoreceptor alters the activity of the histidine kinase, CheA, bound ~300 Å away to the distal cytoplasmic tip of the receptor, to initiate a phosphorylation cascade that controls flagellar rotation. The cytoplasmic domain of the receptor is thought to propagate this signal via changes in dynamics and/or stability, but it is unclear how these changes modulate the kinase activity of CheA. To address this question, we have used hydrogen deuterium exchange mass spectrometry to probe the structure and dynamics of CheA within functional signaling complexes of the Escherichia coli aspartate receptor cytoplasmic fragment, CheA, and CheW. Our results reveal that stabilization of the P4 catalytic domain of CheA correlates with kinase activation. Furthermore, differences in activation of the kinase that occur during sensory adaptation depend on receptor destabilization of the P3 dimerization domain of CheA. Finally, hydrogen exchange properties of the P1 domain that bears the phosphorylated histidine identify the dimer interface of P1/P1' in the CheA dimer and support an ordered sequential binding mechanism of catalysis, in which dimeric P1/P1' has productive interactions with P4 only upon nucleotide binding. Thus stabilization/destabilization of domains is a key element of the mechanism of modulating CheA kinase activity in chemotaxis, and may play a role in the control of other kinases.

Suppressed Size Effect in Nanopillars with Hierarchical Microstructures Enabled by Nanoscale Additive Manufacturing.

Studies on mechanical size effects in nanosized metals unanimously highlight both intrinsic microstructures and extrinsic dimensions for understanding size-dependent properties, commonly focusing on strengths of uniform microstructures, e.g., single-crystalline/nanocrystalline and nanoporous, as a function of pillar diameters, D. We developed a hydrogel infusion-based additive manufacturing (AM) technique using two-photon lithography to produce metals in prescribed 3D-shapes with ∼100 nm feature resolution. We demonstrate hierarchical microstructures of as-AM-fabricated Ni nanopillars (D ∼ 130-330 nm) to be nanoporous and nanocrystalline, with d ∼ 30-50 nm nanograins subtending each ligament in bamboo-like arrangements and pores with critical dimensions comparable to d. In situ nanocompression experiments unveil their yield strengths, σ, to be ∼1-3 GPa, above single-crystalline/nanocrystalline counterparts in the D range, a weak size dependence, σ ∝ D-0.2, and localized-to-homogenized transition in deformation modes mediated by nanoporosity, uncovered by molecular dynamics simulations. This work highlights hierarchical microstructures on mechanical response in nanosized metals and suggests small-scale engineering opportunities through AM-enabled microstructures.

Mpox diagnostics: Review of current and emerging technologies.

Mpox is a zoonotic disease caused by monkeypox virus (MPXV) from the Orthopoxvirus genus. Unprecedented transmission events have led to more than 70 000 cases reported worldwide by October 2022. The change in mpox epidemiology has raised concerns of its ability to establish endemicity beyond its traditional geographical locations. In this review, we discuss the current understanding of mpox virology and viral dynamics that are relevant to mpox diagnostics. A synopsis of the traditional and emerging laboratory technologies useful for MPXV detection and in guiding "elimination" strategies is outlined in this review. Importantly, development in MPXV genomics has rapidly advanced our understanding of the role of viral evolution and adaptation in the current outbreak.

Intra- and interhost genomic diversity of monkeypox virus.

The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high-incidence nations in Europe and the Americas, with a paucity of data from South-East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within-host diversity and instances of co-infection, and highlight further examples of structural variation and apolipoprotein B editing complex-driven micro-evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus' evolutionary trajectory throughout the mpox outbreak.

Neighborhood disadvantage and health-related quality of life in pediatric epilepsy.

INTRODUCTION: While several demographic and epilepsy-specific characteristics are associated with diminished HRQoL in children and adolescents with epilepsy, prior investigations have failed to incorporate and address the influence of broader social contextual factors on functional outcomes. To address this gap, the purpose of the current study was to investigate the role of neighborhood disadvantage on HRQoL, including the extent to which familial and seizure-specific risk factors are impacted. METHODS: Data included parental ratings on the Quality of Life in Childhood Epilepsy (QOLCE) questionnaire for 135 children and adolescents with epilepsy, and the Area Deprivation Index (ADI) to measure neighborhood disadvantage. Bivariate correlations were conducted to identify significant associations with neighborhood disadvantage, followed by a three-stage hierarchical multiple regression to predict HRQoL. Follow-up binary logistic regressions were used to determine the risk conferred by neighborhood disadvantage on sociodemographic, seizure-specific, and HRQoL factors. RESULTS: Moderate associations between neighborhood disadvantage and familial factors, including parental psychiatric history and Medicaid insurance, were identified, while disadvantage and greater seizure frequency were marginally associated. Neighborhood disadvantage independently predicted HRQoL, and was the sole significant predictor of HRQoL when familial factors were incorporated. Children with epilepsy living in disadvantaged areas were four times more likely to have diminished HRQoL, five times more likely to live with a parent with a significant psychiatric history, and four times more likely to reside with a family receiving Medicaid insurance. CONCLUSIONS: These results highlight the importance of identifying high-risk groups, as the cumulative burden of social context, familial factors, and seizure-specific characteristics contribute to lower HRQoL in pediatric epilepsy which disproportionately affects patients from lower-resourced backgrounds. Potentially modifiable factors such as parental psychiatric status exist within the child's environment, emphasizing the importance of a whole-child approach to patient care. Further exploration of disadvantage in this population is needed to better understand these relationships over time.

Emergence of Attenuated Measles Illness Among IgG-positive/IgM-negative Measles Cases: Victoria, Australia, 2008-2017.

BACKGROUND: Waning measles immunity among vaccinated individuals may result in an attenuated illness. This study compares the epidemiological, clinical, and laboratory profile of measles cases with waning immunity with other measles cases. METHODS: Polymerase chain reaction-positive (+) measles cases notified to Victoria's Department of Health and Human Services from 2008 to 2017 with immunoglobulin (Ig) M and IgG tested at diagnosis were classified according to serology at diagnosis: IgG negative (-) (nonimmune), IgM+/IgG+ (indeterminate), or IgM-/IgG+ (waning immunity). RESULTS: Between 2008 and 2017, 297 measles cases were notified, of whom 190 (64%) were included; 151 of 190 (79%) were nonimmune at diagnosis, 26 (14%) were indeterminate, and 13 (7%) had waning immunity. Between 2008-2013 and 2014-2017, the proportion of cases with waning immunity increased from 0 of 87 (0%) to 13 of 103 (13%) (P < .001) and the diagnostic sensitivity of initial IgM fell from 93% to 81% (P = .012), respectively. Seven (54%) waning immunity cases reported receiving measles-containing vaccines; 1 case had 2 documented doses. Compared with nonimmune and indeterminate cases, waning immunity cases were more likely to be male; less likely to report fever, coryza, and cough; and had lower burden of virus (higher cycle threshold values). Waning immunity cases had higher IgG titers than indeterminate cases (mean optical density values, 1.96 vs 0.71; P = .004). Onward transmission from 1 waning immunity case was documented. CONCLUSIONS: Waning immunity among measles cases, associated with secondary vaccine failure and modified clinical illness, is emerging in Victoria with transmission potential.

Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia.

OBJECTIVES: To describe the first isolation and sequencing of SARS-CoV-2 in Australia and rapid sharing of the isolate. SETTING: SARS-CoV-2 was isolated from a 58-year-old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID-19 (the illness caused by SARS-CoV-2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS-CoV-2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS-CoV-2 genomes. Within 24 hours of isolation, the first Australian SARS-CoV-2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS-CoV-2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

Ganciclovir-resistant post-transplant cytomegalovirus infection due to combined deletion mutation at codons 595-596 of the UL97 gene.

The development of antiviral-resistant cytomegalovirus (CMV) infection complicates the management of transplant recipients. We describe the case of a 65-year-old male who developed CMV disease on valganciclovir prophylaxis (donor CMV IgG positive, recipient CMV IgG indeterminate) 30 days after combined liver-kidney transplantation for alcoholic cirrhosis and hepato-renal syndrome. After an initial complete response to treatment dose oral valganciclovir, he developed recurrent CMV viraemia. Resistance testing revealed a UL97 mutation with in-frame deletions of codons 595-596. He was treated successfully with foscarnet and reduction in immunosuppression. This mutation has not been described previously and was suspected to confer ganciclovir resistance. Ganciclovir resistance occurs most commonly due to mutations in the UL97 or UL54 genes, which encode a protein kinase and a DNA polymerase, respectively. The UL97-encoded protein kinase phosphorylates ganciclovir to ganciclovir triphosphate, which competitively inhibits viral replication. Mutations in the UL97 gene are typically point mutations or deletions. We describe a new mutation, del595-596 in the CMV UL97 gene, occurring in the context of clinical treatment failure with standard and double-dose ganciclovir, and successful virological control achieved with foscarnet. This mutation is likely to result in ganciclovir resistance, although recombinant phenotyping is required for confirmation.

Utility of a Stressed Single Nucleotide Polymorphism (SNP) Real-Time PCR Assay for Rapid Identification of Measles Vaccine Strains in Patient Samples.

Rapid differentiation of wild-type measles virus from measles vaccine strains is crucial during a measles outbreak and in a measles elimination setting. A real-time reverse transcription-PCR (rRT-PCR) for the rapid detection of measles vaccine strains was developed with high specificity and sensitivity equivalent to that of traditional measles genotyping methods. The "stressed" minor groove binder-TaqMan probe design approach achieves specificity to vaccine strains only, without compromising sensitivity. This assay, without requiring sequence genotyping, has proved to be extremely useful in outbreak settings for over 4 years at the Regional Measles Reference Laboratory for the Western Pacific Region.

The role of human rhinovirus (HRV) species on asthma exacerbation severity in children and adolescents.

OBJECTIVE: It is recognized that human rhinovirus (HRV) infection is an important factor in asthma exacerbations requiring hospitalization in children. However, previous studies have disagreed on the differential impact of various HRV species. We sought to assess the impact of HRV species on the severity of asthma exacerbations in children and adolescents. We also examined whether the effect of HRV species on severity was modified by age and gender. METHODS: Virus strain was determined for 113 children with HRV detectable at the time of admission for asthma exacerbation. Patient characteristics were collected on admission and exacerbation severity was scored using several validated scales. RESULTS: HRV species by itself was not associated with moderate/severe vs. mild exacerbations. Boys with HRV-C infections were more likely (OR: 3.7, 95% CI: 1.2-13.4) to have a moderate/severe exacerbation than girls with HRV-C (p = 0.04 for interaction term). Higher odds were observed in younger boys (3 years old: OR: 9.1, 95% CI: 1.8-47.1 vs 5 years old: OR: 3.3, 95% CI: 0.9-11.8 vs 7 years old: OR: 1.2, 95% CI: 0.2-6.6). In contrast, children with HRV-C infection and sensitized to pollen during the pollen season were less likely to have moderate/severe exacerbations (p = 0.01 for the interaction term). CONCLUSION: Acute asthma exacerbations are more likely to be moderate/severe in boys under 5 years of age who had HRV-C infection on admission. The opposite was found in children with sensitization to pollen during pollen season.

Comparison of cardiac output optimization with an automated closed-loop goal-directed fluid therapy versus non standardized manual fluid administration during elective abdominal surgery: first prospective randomized controlled trial.

An intraoperative automated closed-loop system for goal-directed fluid therapy has been successfully tested in silico, in vivo and in a clinical case-control matching. This trial compared intraoperative cardiac output (CO) in patients managed with this closed-loop system versus usual practice in an academic medical center. The closed-loop system was connected to a CO monitoring system and delivered automated colloid fluid boluses. Moderate to high-risk abdominal surgical patients were randomized either to the closed-loop or the manual group. Intraoperative final CO was the primary endpoint. Secondary endpoints were intraoperative overall mean cardiac index (CI), increase from initial to final CI, intraoperative fluid volume and postoperative outcomes. From January 2014 to November 2015, 46 patients were randomized. There was a lower initial CI (2.06 vs. 2.51 l min-1 m-2, p = 0.042) in the closed-loop compared to the control group. No difference in final CO and in overall mean intraoperative CI was observed between groups. A significant relative increase from initial to final CI values was observed in the closed-loop but not the control group (+ 28.6%, p = 0.006 vs. + 1.2%, p = 0.843). No difference was found for intraoperative fluid management and postoperative outcomes between groups. There was no significant impact on the primary study endpoint, but this was found in a context of unexpected lower initial CI in the closed-loop group.Trial registry number ID-RCB/EudraCT: 2013-A00770-45. ClinicalTrials.gov Identifier NCT01950845, date of registration: 17 September 2013.

Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment.

The global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.

Low interim influenza vaccine effectiveness, Australia, 1 May to 24 September 2017.

In 2017, influenza seasonal activity was high in the southern hemisphere. We present interim influenza vaccine effectiveness (VE) estimates from Australia. Adjusted VE was low overall at 33% (95% confidence interval (CI): 17 to 46), 50% (95% CI: 8 to 74) for A(H1)pdm09, 10% (95% CI: -16 to 31) for A(H3) and 57% (95% CI: 41 to 69) for influenza B. For A(H3), VE was poorer for those vaccinated in the current and prior seasons.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Interparental Coercive Control and Child and Family Outcomes: A Systematic Review.

Coercive control (CC) is a core facet of intimate partner violence (IPV) and involves asserting power, dominance, and control over another person. Although the adverse impacts of childhood exposure to interparental IPV have been well documented, the outcomes of childhood exposure to interparental CC have not been systematically examined. This study aimed to address this gap by reviewing available empirical evidence on interparental CC and child and family outcomes. Articles were identified by searching electronic databases using keywords relating to CC, children and parents, and child wellbeing outcomes. The final review included 51 studies that reported on adverse outcomes pertaining to parenting and family relationships (k = 29), child internalizing and externalizing problems (k = 7), social-emotional development (k = 5), and physical/health development (k = 17). Specifically, studies reported that CC was associated with increased parental psychopathology, poorer family functioning, harsher parenting and higher levels of child abuse, strained parent-child relationships, children used as tools and co-victims of CC, increased risk of child internalizing and externalizing problems, limited socializing opportunities, increased bullying, poorer perinatal outcomes, limited access to healthcare, and increased risk of child mortality. Evidence identified CC as a unique contributor to adverse child wellbeing outcomes, independent of exposure to IPV more broadly. Results indicated that the impacts of childhood exposure to CC are complex, far reaching, and, in some cases, devastating. The limitations of the findings, as well as implications for practice, policy, and research are discussed.

Approaching Standardization: Mechanical Material Testing of Macroscopic Two-Photon Polymerized Specimens.

Two-photon polymerization (2PP) is becoming increasingly established as additive manufacturing technology for microfabrication due to its high-resolution and the feasibility of generating complex parts. Until now, the high resolution of 2PP is also its bottleneck, as it limited throughput and therefore restricted the application to the production of microparts. Thus, mechanical properties of 2PP materials can only be characterized using nonstandardized specialized microtesting methods. Due to recent advances in 2PP technology, it is now possible to produce parts in the size of several millimeters to even centimeters, finally permitting the fabrication of macrosized testing specimens. Besides suitable hardware systems, 2PP materials exhibiting favorable mechanical properties that allow printing of up-scaled parts are strongly demanded. In this work, the up-scalability of three different photopolymers is investigated using a high-throughput 2PP system and low numerical aperture optics. Testing specimens in the cm-range are produced and tested with common or even standardized material testing methods available in conventionally equipped polymer testing labs. Examples of the characterization of mechanical, thermo-mechanical, and fracture properties of 2PP processed materials are shown. Additionally, aspects such as postprocessing and aging are investigated. This lays a foundation for future expansion of the 2PP technology to broader industrial application.

Non-SARS-CoV-2 respiratory viral detection and whole genome sequencing from COVID-19 rapid antigen test devices: a laboratory evaluation study.

BACKGROUND: There has been high uptake of rapid antigen test device use for point-of-care COVID-19 diagnosis. Individuals who are symptomatic but test negative on COVID-19 rapid antigen test devices might have a different respiratory viral infection. We aimed to detect and sequence non-SARS-CoV-2 respiratory viruses from rapid antigen test devices, which could assist in the characterisation and surveillance of circulating respiratory viruses in the community. METHODS: We applied archival clinical nose and throat swabs collected between Jan 1, 2015, and Dec 31, 2022, that previously tested positive for a common respiratory virus (adenovirus, influenza, metapneumovirus, parainfluenza, rhinovirus, respiratory syncytial virus [RSV], or seasonal coronavirus; 132 swabs and 140 viral targets) on PCR to two commercially available COVID-19 rapid antigen test devices, the Panbio COVID-19 Ag Rapid Test Device and Roche SARS-CoV-2 Antigen Self-Test. In addition, we collected 31 COVID-19 rapid antigen test devices used to test patients who were symptomatic at The Royal Melbourne Hospital emergency department in Melbourne, Australia. We extracted total nucleic acid from the device paper test strips and assessed viral recovery using multiplex real-time PCR (rtPCR) and capture-based whole genome sequencing. Sequence and genome data were analysed through custom computational pipelines, including subtyping. FINDINGS: Of the 140 respiratory viral targets from archival samples, 89 (64%) and 88 (63%) were positive on rtPCR for the relevant taxa following extraction from Panbio or Roche rapid antigen test devices, respectively. Recovery was variable across taxa: we detected influenza A in nine of 18 samples from Panbio and seven of 18 from Roche devices; parainfluenza in 11 of 20 samples from Panbio and 12 of 20 from Roche devices; human metapneumovirus in 11 of 16 from Panbio and 14 of 16 from Roche devices; seasonal coronavirus in eight of 19 from Panbio and two of 19 from Roche devices; rhinovirus in 24 of 28 from Panbio and 27 of 28 from Roche devices; influenza B in four of 15 in both devices; and RSV in 16 of 18 in both devices. Of the 31 COVID-19 devices collected from The Royal Melbourne Hospital emergency department, 11 tested positive for a respiratory virus on rtPCR, including one device positive for influenza A virus, one positive for RSV, four positive for rhinovirus, and five positive for SARS-CoV-2. Sequences of target respiratory viruses from archival samples were detected in 55 (98·2%) of 56 samples from Panbio and 48 (85·7%) of 56 from Roche rapid antigen test devices. 98 (87·5%) of 112 viral genomes were completely assembled from these data, enabling subtyping for RSV and influenza viruses. All 11 samples collected from the emergency department had viral sequences detected, with near-complete genomes assembled for influenza A and RSV. INTERPRETATION: Non-SARS-CoV-2 respiratory viruses can be detected and sequenced from COVID-19 rapid antigen devices. Recovery of near full-length viral sequences from these devices provides a valuable opportunity to expand genomic surveillance programmes for public health monitoring of circulating respiratory viruses. FUNDING: Australian Government Medical Research Future Fund and Australian National Health and Medical Research Council.