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Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam 2 Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4 + T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam 2 Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses. Current vaccines against SARS-CoV-2 reduce mortality but are less effective in preventing infection. Here the authors show that intranasal vaccination with a subunit vaccine including an TLR2-stimulating adjuvant induces strong neutralising antibody and T-cell responses against SARS-CoV-2 in the lungs that protect against infection.

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation. TANK-binding kinase-1 (TBK1) and its homologue IκB kinase-ε (IKKε) are critical in the induction of the interferon response and the response to infection by pathogens. Here the authors show that pharmacological targeting of TBK1 AND IKKε reduces the immunopathology seen in a murine model of SARS-COV-2 infection.

This short communication uses numerical continuation to highlight the existence of an isola in a simple one‐degree‐of‐freedom harmonically forced feedback system with actuator rate limiting as its only nonlinear element. It was found that the isola (1) contains only rate‐limited responses, (2) merges with the main branch when the forcing amplitude is sufficiently large, and (3) includes stable solutions that create a second attractor in regions where rate limiting is not expected. Furthermore, the isola is composed of two solutions for a given forcing frequency. These solutions have the same amplitudes in the state (pitch rate) projection; however, they have distinct phases, and their amplitudes are also distinct when projected onto the integrator state in the controller. The rich dynamics observed in such a simple example underlines the impact of rate limiting on feedback systems. Specifically, the combination of feedback and rate limiting can create detrimental dynamics that is hard to predict and requires careful analysis.

Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

Injected COVID-19 vaccines protect against severe disease, but do not induce robust mucosal immune responses. Nasal vaccines offer the advantage of local immunity to block viral infection and transmission. Previously we showed immunization of a Pam 2 Cys-adjuvanted SARS-CoV-2 vaccine to the upper and lower respiratory tracts (URT/LRT) induced protective immune responses in the lungs. However, URT/LRT immunization is not representative of nasal vaccines for clinical use that exclusively target the URT. Here, we show that delivery to only the URT with Pam 2 Cys and spike protein effectively induced strong SARS-CoV-2 specific immune responses in the nasal mucosa. When delivered in a low volume so that vaccine exposure was limited to the URT, Pam 2 Cys/spike protein induced local SARS-CoV-2-specific Th17 cells and neutralizing antibodies to a similar level to inhaled vaccination reaching both the URT and LRT. We compared URT versus URT/LRT delivery as booster vaccinations following parenteral immunization and found that URT vaccination concentrated the immune response to the URT rather than the lungs. Importantly, URT immunization or boosting induced sterilizing immunity in K18-hACE2 mice challenged with homologous SARS-CoV-2. Thus, booster vaccination to the URT alone with Pam 2 Cys/spike achieved robust nasal immunity against SARS-CoV-2 and is a promising strategy for clinical development.

Background Stillbirths and neonatal deaths are devastating events for both parents and clinicians and are global public health concerns. Careful clinical management after these deaths is required, including appropriate investigation and assessment to determine cause (s) to prevent future losses, and to improve bereavement care for families. An educational programme for health care professionals working in maternal and child health has been designed to address these needs according to the Perinatal Society of Australia and New Zealand Guideline for Perinatal Mortality: IMproving Perinatal mortality Review and Outcomes Via Education (IMPROVE). The programme has a major focus on stillbirth and is delivered as six interactive skills-based stations. We aimed to determine participants’ pre- and post-programme knowledge of and confidence in the management of perinatal deaths, along with satisfaction with the programme. We also aimed to determine suitability for international use. Methods The IMPROVE programme was delivered to health professionals in maternity hospitals in all seven Australian states and territories and modified for use internationally with piloting in Vietnam, Fiji, and the Netherlands (with the assistance of the International Stillbirth Alliance, ISA). Modifications were made to programme materials in consultation with local teams and included translation for the Vietnam programme. Participants completed pre- and post-programme evaluation questionnaires on knowledge and confidence on six key components of perinatal death management as well as a satisfaction questionnaire. Results Over the period May 2012 to May 2015, 30 IMPROVE workshops were conducted, including 26 with 758 participants in Australia and four with 136 participants internationally. Evaluations showed a significant improvement between pre- and post-programme knowledge and confidence in all six stations and overall, and a high degree of satisfaction in all settings. Conclusions The IMPROVE programme has been well received in Australia and in three different international settings and is now being made available through ISA. Future research is required to determine whether the immediate improvements in knowledge are sustained with less causes of death being classified as unknown, changes in clinical practice and improvement in parents’ experiences with care. The suitability for this programme in low-income countries also needs to be established.

Based on bifurcation theory, nonlinear frequency response analysis is a recent development in the field of flight dynamics studies. Here, we consider how this method can be used to inform us on how to devise the control input such that the system transitions from an undesirable equilibrium solution—an aircraft deep stall solution in our case—to a desirable solution. We show that it is still possible to induce a large-amplitude oscillation via harmonic forcing of the pitch control device and escape the otherwise unrecoverable deep stall, despite very little control power available in such a high angle-of-attack flight condition. The forcing frequencies that excite these resonances are reflected as asymptotically unstable solutions using bifurcation analysis and Floquet theory. Due to the softening behaviour observed in the frequency response, these unstable (divergent) solutions have slightly lower frequencies than the value predicted using linear analysis. Subharmonic resonances are also detected, which are reflected in the time-domain unforced responses. These nonlinear phenomena show strong dependency on the forcing/perturbation amplitude and result in complex dynamics that can impede recovery if the existing procedures are followed. The proposed method is shown to be a useful tool for nonlinear flight dynamics analysis as well as to complement the rather thin literature on deep stall analysis—a topic of relevance for recent research on unconventional landing techniques in unmanned aerial vehicles. A full description of the aircraft model used, the unstable F-16 fighter jet, is provided in the appendix.

Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies. Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects. These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.

Background Congenital adrenal hyperplasia (CAH) (OMIM #201910) is a complex disease most often caused by pathogenic variant of the CYP21A2 gene. We have designed an efficient multistep approach to diagnose and classify CAH cases due to CYP21A2 variant and to study the genotype‐phenotype relationship. Methods A large cohort of 212 Vietnamese patients from 204 families was recruited. We utilized Multiplex Ligation‐dependent Probe Amplification to identify large deletion or rearrangement followed by complete gene sequencing of CYP21A2 to map single‐nucleotide changes and possible novel variants. Results Pathogenic variants were identified in 398 out of 408 alleles (97.5%). The variants indexed span across most of the CYP21A2 gene regions. The most common genotypes were: I2g/I2g (15.35%); Del/Del (14.4%); Del/I2g (10.89%); p.R356W/p.R356W (6.44%); and exon 1–3 del/exon 1–3 del (5.44%). In addition to the previously characterized and documented variants, we also discovered six novel variants which were not previously reported, in silico tools were used to support the pathogenicity of these variants. Conclusion The result will contribute in further understanding the genotype‐phenotype relationship of CAH patients and to guide better treatment and management of the affected. This is a mutation study in a large cohort of 212 patients with congenital adrenal hyperplasia (CAH) in Vietnam, in which we discovered six novel disease causing variants of CYP21A2. This is the first mutation study of CAH in Vietnam and the result should help in further understanding the genotype‐phenotype relationship of CAH and will guide better treatment and management of the disorder.