No abstract

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

Due to differences in human and murine angiotensin converting enzyme 2 (ACE-2) receptor, initially available SARS-CoV-2 isolates could not infect mice. Here we show that serial passaging of USA-WA1/2020 strain in mouse lungs results in “mouse-adapted” SARS-CoV-2 (MA-SARS-CoV-2) with mutations in S, M, and N genes, and a twelve-nucleotide insertion in the S gene. MA-SARS-CoV-2 infection causes mild disease, with more pronounced morbidity depending on genetic background and in aged and obese mice. Two mutations in the S gene associated with mouse adaptation (N501Y, H655Y) are present in SARS-CoV-2 variants of concern (VoCs). N501Y in the receptor binding domain of viruses of the B.1.1.7, B.1.351, P.1 and B.1.1.529 lineages (Alpha, Beta, Gamma and Omicron variants) is associated with high transmissibility and allows VoCs to infect wild type mice. We further show that S protein mutations of MA-SARS-CoV-2 do not affect neutralization efficiency by human convalescent and post vaccination sera.

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono-and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and Ras G12V proteins when incubated with JAK2 or SRC kinases but not of Ras Y4F or Ras Y4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of Ras G12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.

SARS-CoV-2 is the cause of one of the largest noninfluenza pandemics of this century. This exceptional public health crisis highlights the urgent need for better understanding of the correlates of protection from infection and severe COVID-19.

One of the emerging trends in contemporary healthcare is the shift in surgical resources to the outpatient setting coupled with institutional focus of reducing inpatient length of stay to facilitate cost containment. Through a multi-center retrospective review of operatively treated ankle fractures, we sought to calculate the actual financial cost of each procedure as well as to compare the economics of performing outpatient versus inpatient surgery. Additionally, we sought to determine whether the surgery location (inpatient and outpatient) was associated with specific patient demographics, medical comorbidities, or surgeon practice patterns. Methods: A multi-center retrospective comparative study of 240 surgically treated ankle fractures over a two-year period was performed. Two tertiary care hospitals and their affiliated ambulatory surgery centers were included in the study. Patient selection was based on Current Procedural Terminology codes while exclusion criteria included pediatric patient, open trauma, distal tibia pilon fracture, or history of prior ankle fracture. The total direct cost of each surgery was calculated including categorized charges for room and board, pharmacy, rehabilitation, radiology, surgical implant materials, and surgeon professional fees. Patient age, medical co-morbidities, presence of poly-trauma, ordering of ankle CT-scan, and fellowship training of the orthopaedic surgeon were also evaluated in the study. The chi-square test or Fisher's exact test was used to compare inpatients and outpatients for each variable. Results: 142 inpatient and 98 outpatient ankle fracture surgeries were performed. Median length of stay was 5 days for inpatients and the mean total direct cost was $11,466 for each inpatient case with room and board charges averaging $2,694. The mean total direct cost for each outpatient procedure was $3,111. Regarding patient demographics, statistically significant higher percentages were recorded among inpatients in the following groups: age 65 years or older (p < 0.0003), hypertension (p < 0.0230), presence of poly-trauma (p < 0.0149) and ordering of ankle CT-scan (p < 0.0001). 84% of ankle fracture surgeries performed by foot and ankle surgeons were outpatient procedures while 71% of ankle fracture surgeries performed by orthopaedic trauma surgeons were inpatient procedures. Conclusion: Our data shows that with 5 day median length of stay for the hospitalized patient group, the average total cost for inpatient ankle fracture surgery was nearly four times higher and $8,000 more than the total cost for outpatient ankle fracture surgery. Increased patient age and other specific medical co-morbidities were statistically linked with inpatient admission. In this multi-center study, foot and ankle surgeons were more likely than trauma surgeons to perform outpatient ankle fracture surgery. Healthcare institutions may realize substantial practice management cost savings by shifting ankle fracture surgery to the outpatient setting.