Utility of tofacitinib beyond autoimmunity

Objectives: This study was conducted to describe the use of tofacitinib in severe and critical Coronavirus disease -2019 (COVID-19), and to explore the association of drug initiation time with survival. Method(s): This was a retrospective chart review of inpatients with severe or critical COVID-19 at a tertiary care hospital, who received generic tofacitinib for at least 48 hours. The baseline demographics, comorbidities, treatment, adverse effects and outcomes (i.e. mortality at day 28) were analysed. The severity of COVID-19 was categorised as per WHO classification. Patients were further grouped based on median duration of symptomatic illness prior to tofacitinib administration, as early or late initiation groups. Result(s): Forty-one patients [(85.4% males), mean age 52.9 +/- 12.5 years], were studied. 65.9% (n = 27) of patients had severe COVID-19, while 34.1% (n = 14) were critically ill. Death occurred in 36.6% patients (n = 15). The median time to prescription of tofacitinib was 13 (9.50, 16.0) days of symptom onset. Tofacitinib was initiated early (8-13 days) in 56.1% of patients (n = 23), while the remaining received it beyond day 14 of symptom onset (late initiation group). The proportion of survivors was significantly higher in the early initiation group (21/23, 91.3%) compared to the late group (5/18, 27.8%) (P < 0.0001). Among severe COVID-19 patients, 100% and 62.5% of the patients were survivors among early and late initiation groups respectively (P < 0.01). In the critical COVID-19 patients, 50% were alive on day 28 in the early group while all died in the hospital in the late initiation group (P = 0.06). Multivariate logistic regression adjusted for age, presence of diabetes mellitus (DM) and illness duration prior to hospitalisation demonstrated higher odds of survival (AOR-19.3, 95% C.I. 2.57, 145.2) in the early initiation group, compared to the late initiation group. Conclusion(s): Early initiation of tofacitinib in severe and critical COVID-19 has potential to improve survival odds. (Table Presented).

Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

Passive immunization with equine RBD-specific Fab protects K18-hACE2-mice against Alpha or Beta variants of SARS-CoV-2.

Emergence of variants of concern (VOC) during the COVID-19 pandemic has contributed to the decreased efficacy of therapeutic monoclonal antibody treatments for severe cases of SARS-CoV-2 infection. In addition, the cost of creating these therapeutic treatments is high, making their implementation in low- to middle-income countries devastated by the pandemic very difficult. Here, we explored the use of polyclonal EpF(ab')2 antibodies generated through the immunization of horses with SARS-CoV-2 WA-1 RBD conjugated to HBsAg nanoparticles as a low-cost therapeutic treatment for severe cases of disease. We determined that the equine EpF(ab')2 bind RBD and neutralize ACE2 receptor binding by virus for all VOC strains tested except Omicron. Despite its relatively quick clearance from peripheral circulation, a 100µg dose of EpF(ab')2 was able to fully protect mice against severe disease phenotypes following intranasal SARS-CoV-2 challenge with Alpha and Beta variants. EpF(ab')2 administration increased survival while subsequently lowering disease scores and viral RNA burden in disease-relevant tissues. No significant improvement in survival outcomes or disease scores was observed in EpF(ab')2-treated mice challenged using the Delta variant at 10µg or 100µg doses. Overall, the data presented here provide a proof of concept for the use of EpF(ab')2 in the prevention of severe SARS-CoV-2 infections and underscore the need for either variant-specific treatments or variant-independent therapeutics for COVID-19.

SARS-CoV-2 receptor binding domain displayed on HBsAg virus-like particles elicits protective immunity in macaques.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain-hepatitis B surface antigen virus-like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>104) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log10) and nasal mucosa (~2.9 log10) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii (preprint)

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

COVID 19 awareness and its impact on periodontal practice among dental care professionals in southern states of india-a cross sectional survey

Background: COVID 19 pandemic has nevertheless gripped our nation with its unshackled spread, varied disease characterization posing a real threat to health care workers and a high risk to dental professionals necessitating the publishing of guidelines to help dentists continue to provide optimum health services with safety. This cross-sectional survey aims to assess the awareness of the disease, knowledge of infection control and impact of COVID-19 pandemic on periodontal practice. Methods: The survey was conducted among 250 participants. The study instrument was a structured questionnaire that was grouped into four sections, namely demographic characteristics;awareness of the COVID pandemic, employment of infection control measures in combating COVID-19 pandemic;and the impact of COVID on periodontal practice. Statistical analysis was carried out using SPSS software and the data was presented as percentages. Conclusion: A total of 206 responses were considered as complete submissions which included 71.1%from the red zone, 12.5 % from the orange zone and 16.4% from the green zone. In conclusion, our study revealed that periodontists from Southern states of India were well acquainted regarding the pandemic. It is recommended that dentists should update themselves with the changing norms and adhere to follow national and international guidelines.