Comparison of upper and lower airway expression of SARS-COV-2 receptors in mild allergic asthmatics

Background: SARS-CoV-2 virus infects host cells through ACE2 and TMPRSS2 receptors. Protein levels of ACE2 and TMPRSS2 have not been assessed in allergic airways. Method(s): We collected biopsies of endobronchial tissue from steroid-naive mild allergic asthmatics (AA n=23) and non-asthmatic controls (NA n=11), and inferior nasal turbinate tissue from AA with allergic rhinitis (AR n=8) and nonAA/AR controls (NR n=5). Tissue was immune-stained for SARS-CoV-2 receptor ACE2 and surface protein TMPRSS2. The number of immuno-positive cells in epithelium and laminae propria was expressed per mm2 of tissue. Result(s): The number of cells expressing ACE2 was higher in AA endobronchial tissue compared to NA control and AR nasal tissue. TMPRSS2 was higher in AR nasal tissue compared to NR control, and higher in control NA endobronchial tissue versus control NR nasal tissue. Co-expression of ACE2+TMPRSS2 was higher in AA endobronchial tissue versus NA control and trending higher in AR nasal tissue versus NR control (p=0.08). Conclusion(s): Overall, ACE2 is more highly expressed in endobronchial tissue versus nasal tissue, suggesting SARS-CoV-2 may more readily infect lower versus upper airways. It is unknown whether the higher expression of ACE2 and ACE2+TMPRSS2 observed in the airways of mild allergic asthmatic donors versus control donors translates to higher susceptibility to infection.

Reduced ACE2 and TMPRSS2 immunopositive bronchial cells in asthmatics after inhaled allergen challenge

Rationale: The incidence of SARS-CoV-2 infection and the impact of corticosteroid treatment in patients with symptomatic airway disease has been a concern. We examined airway expression of SARS-CoV-2 receptors following allergen challenge and steroid intervention in asthmatic patients. Method(s): From steroid-naive mild allergic asthmatic (AA n=23) we collected endobronchial biopsies pre and 24hr post allergen inhalation challenge (AIC). In a subset of AA with allergic rhinitis (AR n=8) we collected inferior nasal turbinate biopsies pre and 24hr post-nasal allergen challenges (NAC) after placebo treatment or after 21 days of 22 mg BID triamcinolone nasal spray. FEV1 and PNIF expressed as % fall from baseline quantified the early (ER, 0-2h) and late (LR, 3-7h) airway responses post challenge. Epithelium and laminae propria were immunostained for ACE2 and TMPRSS2 and expressed as # cells/mm2. Result(s): AIC reduced FEV1 (31% ER, 19% LR) and the number of bronchial cells immunopositive for ACE2, TMPRSS2 and double positive for ACE2/TMPRSS2 (P=0.0002, P=0.04, P=0.02, respectively). The PNIF reduction by NAC (69% ER, 49% LR) was attenuated by triamcinolone (31% ER, 18% LR), but without changes in ACE2 or TMPRSS2 in nasal tissue after NAC or steroid treatment (all P>0.05). In the nasal tissue, significantly fewer cells expressed ACE2 compared to bronchi (P=0.007). Conclusion(s): ACE2 and TMPRSS2 expression in bronchial tissue is reduced in the T2 microenvironment post allergen challenge, however it is unknown if this protects lower airways from SARS-CoV-2 infection. Low expression of ACE2 and TMPRSS2 in nasal tissue made it difficult to determine the effects of NAC or steroid.

RISING TO THE CHALLENGE: ORTHOGERIATRIC SERVICE IMPROVEMENT AND COVID-19

Background: Nationally agreed hip fracture standards have contributed to the improvement of outcomes in hip fracture patients. In 2020, our hospital was awarded “The Golden Hip” for achieving highest compliance with Irish Hip Fracture Standards (IHFS) nationally for 2019. Methods: Data from the Irish Hip Fracture Database (IHFD)was retrospectively analysed to assess our performance in 2020 versus 2019 in hip fracture patients over sixty. Multiple quality improvement interventions were put in place throughout 2019 to ensure improvement in IHFS1-6 compliance: Creation of the Hip Fracture Pathway Subgroup, IHFS 1 Breaches Audit, Orthogeriatric input at Orthopaedic inductions, weekly Multi-disciplinary Team meetings, a Nutritional Hip Fracture Pathway and addition of the Fracture Liaison Service Advanced Nurse Practitioner. Results: There were 239 hip fracture patients in 2020 vs 249 in 2019. IHFS1 compliance improved with the percentage of patients admitted to the Orthopaedic ward within 4 hours increasing to 71% in 2020 from 56% in 2019. There was improvement in IHFS2-time to surgery <48 hours- 66% in 2020 vs 60% in 2019. IHFS3-pressure ulcer rate-was at the national average, 3% in 2020 vs 2% in 2019. IHFS4 (reviewed by a Geriatrician), IHFS5 (received a bone health assessment) and IHFS6 (received a specialised falls assessment) were lower overall;87% in 2020 vs 98% in 2019. For all quarters (Q),43% of patients met all IHFS in our hospital in 2020 vs 32% in 2019, resulting in €90,000 in Best Practice Tariff funding. Conclusion: Lower results for IHFS 4,5 and 6 reflect the arrival of the COVID-19 pandemic which led to redeployment of the Orthogeriatric Service and redeployment of the MDT from end of Q1 to Q3. When services in 2020 were preserved,1 in 2 hip fracture patients met all IHFS, vs 1 in 3 patients in 2019. Despite the pandemic, we continued to achieve the highest level of IHFS compliance nationally, being awarded a second consecutive “Golden Hip” for 2020.

CONTINUING TO ‘BE HIP’: ORTHOGERIATRIC SERVICE IMPROVEMENTS IN 2021

Background: Scotland first demonstrated that adherence to nationally agreed hip fracture standards improve patient survival, reduces the duration of admission, and reduces the need for high dependency care. Our study aims to assess adherence to the Irish Hip Fracture Standards (IHFS) in our hospital for 2021 amidst the COVID-19 pandemic, translating to improved clinical outcomes for our patients. Methods: The IHF database was retrospectively analysed, comparing quarters 1-4 in 2021 with our 2020 results. Results: IHFS1, patient time to the ward < 4hours, was maintained at 67% in 2021 versus 71% overall in 2020. There was improvement in IHFS2, time to surgery within 48 hours, up to 73% in 2021 versus 66% in 2020. IHFS3 was 4% in 2021 versus 3% overall in 2020. Further improvements were noted for IHFS4, with 95% of patients reviewed by a Geriatrician in 2021 versus 87% in 2020. IHFS5 also improved with 97% of patients receiving a bone health assessment in 2021 versus 87% in 2020. Moreover, IHFS6, improved with 97% of patients undergoing a specialised falls assessment in 2021 versus 87% in 2020. Conclusion: The improvement in 2021 figures is reflective of the return of redeployed services during the COVID-19 pandemic inclusive of the Orthogeriatric Service, the Fracture Liaison Service Advanced Nurse Practitioner, the Trauma Co-ordinator, and the specialist Orthopaedic ward complete with its Orthopaedic nurses and Multi-Disciplinary Team, and improved Emergency Department pathways. These continued improvements in the IHFS further emphasise that success is dependent on a team that is joined at the hip

Outpatient free tissue transfer in response to COVID-19-related restrictions

Introduction: Inpatient hospitalization is nearly universal after head and neck reconstruction using free tissue transfer;however, the use of minimal access vascularization techniques and low morbidity flaps have allowed for significantly abbreviated stays without compromising patient outcomes. This experience has led to the management of select patients on an outpatient basis during the COVID-19 pandemic. Method: Due to COVID-related hospital policies restricting inpatient admission for nonemergent and nononcologic surgeries, 2 patients elected for outpatient free tissue transfer for management of mandibular osteoradionecrosis with anterolateral thigh fascia lata rescue flaps. Patient selection, management, and outcomes are presented along with a review of the literature. Results: Two patients, ages 62 and 70 years, with intermediate medical comorbidities and histories of chemoradiation for oropharyngeal cancer presented with progressive osteoradionecrosis refractory to conservative management, including debridement and hyperbaric oxygen therapy. They underwent further mandibular debridement and anterolateral thigh fascia lata free tissue transfer vascularized by minimal access approaches to facial and superficial temporal vessels. Surgical procedures lasted an average of 4.4 hours (range, 3.1-5.4) with placement of a facial Penrose drain that was removed prior to discharge and a leg Jackson-Pratt drain that was removed in clinic the following week. The patients were discharged home the same day (7.3 hours after surgical closure) and the following morning (21.2 hours after surgical closure). They have been followed for an average duration of 53 days (range, 39-67) with no known complications. Conclusion: This case series represents the shortest reported hospital duration for head and neck free tissue transfer. Outpatient status was determined through a combination of case selection, patient decision, and constraints secondary to COVID-19 hospital utilization. This report suggests that outpatient free flap surgery is safe under select circumstances.

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines.