Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology. In collaboration with the International AIDS Vaccine Initiative, Inc. (IAVI), Merck & Co., Inc., Rahway, NJ, USA rapidly advanced an investigational SARS-CoV-2 vaccine based on the recombinant vesicular stomatitis virus (rVSV) platform used for the Ebola vaccine ERVEBO (rVSV∆G-ZEBOV-GP). An rVSV∆G-SARS-CoV-2 vaccine candidate was generated using the SARS-CoV-2 spike protein to replace the VSV G protein. The purification process development for this vaccine candidate was detailed in this paper. Areas were highlighted where the ERVEBO platform process was successfully adopted and where additional measures were needed for the SARS-CoV-2 vaccine candidate. These included: (i) endonuclease addition directly into the bioreactor prior to harvest, (ii) inclusion of a core-shell chromatography step for improved purification, and (iii) incorporation of a terminal, sterile filtration step to eliminate the need for aseptic, closed processing. High infectious virus titers were achieved in Phase 3 clinical drug substance (>108 PFU mL-1 ), and process consistency was demonstrated across four large scale batches that were completed in 6 months from clone selection.

Appropriate use of total parenteral nutrition in children with perforated appendicitis.

BACKGROUND: Total parenteral nutrition (TPN) is often used in children with perforated appendicitis, despite the absence of clear indications. We assessed the validity of specific clinical indications for initiation of TPN in this patient cohort. METHODS: Data were gathered prospectively on duration of nil per os (NPO) status and TPN use in a cohort of children treated under a perforated appendicitis protocol during a 19-month period. TPN was started in the immediate postoperative period in patients who had generalized peritonitis and severe intestinal dilatation at operation, or later per the discretion of the attending surgeon. At discharge, TPN was considered to have been used appropriately, according to consensus guidelines, if the patient was NPO≥7days or received TPN≥5days. RESULTS: During the study period, TPN was initiated in 31 (25.4%) of 122 patients operated for perforated appendicitis. Sixteen (51.6%) received TPN per operative finding indications and 15 (48.4%) for prolonged ileus. The operative indications demonstrated 47% sensitivity, 86% specificity, a positive predictive value (PPV) of 35%, and a negative predictive value (NPV) of 91%, when adherence to TPN consensus guidelines was considered the gold standard. CONCLUSION: Patients without severe intestinal dilatation and generalized peritonitis at operation should not be placed on TPN in the immediate postoperative period. Refinement of selection criteria is necessary to further decrease inappropriate TPN use in children with perforated appendicitis. TYPE OF STUDY: Diagnostic Test. LEVEL OF STUDY: II.

Risk stratification in pediatric perforated appendicitis: Prospective correlation with outcomes and resource utilization.

PURPOSE: Despite a wide spectrum of severity, perforated appendicitis in children is typically considered a single entity in outcomes studies. We performed a prospective cohort study to define a risk stratification system that correlates with outcomes and resource utilization. METHODS: A prospective study was conducted of all children operated for perforated appendicitis between May 2015 and December 2016 at a tertiary free-standing university children's hospital. Surgical findings were classified into one of four grades of perforation: I. localized or contained perforation, II. Contained abscess with no generalized peritonitis, III. Generalized peritonitis with no dominant abscess, IV. Generalized peritonitis with one or more dominant abscesses. All patients were treated on a clinical pathway that involved all points of care from admission to final follow-up. Outcomes and resource utilization measures were analyzed using Fisher's exact test, Kruskal-Wallis test, One-way ANOVA, and logistic regression. RESULTS: During the study period, 122 patients completed treatment, and 100% had documented follow-up at a median of 25days after operation. Grades of perforation were: I, 20.5%; II, 37.7%; III, 10.7%; IV, 31.1%. Postoperative abscesses occurred in 12 (9.8%) of patients, almost exclusively in Grade IV perforations. Hospital stay, duration of antibiotics, TPN utilization, and the incidence of postoperative imaging significantly increased with increasing grade of perforation. CONCLUSION: Outcomes and resource utilization strongly correlate with increasing grade of perforated appendicitis. Postoperative abscesses, additional imaging, and additional invasive procedures occur disproportionately in patients who present with diffuse peritonitis and abscess formation. The current stratification allows risk-adjusted outcome reporting and appropriate assignment of resource burden. LEVEL OF EVIDENCE: I (Prognosis Study).

Standardization of care for pediatric perforated appendicitis improves outcomes.

BACKGROUND: The treatment of perforated appendicitis in children is characterized by significant variability in care, morbidity, resource utilization, and outcomes. We prospectively studied how minimization of care variability affects outcomes. METHODS: A clinical pathway for perforated appendicitis, in use for three decades, was further standardized in May 2015 by initiation of a disease severity classification, refinement of discharge criteria, standardization of the operation, and establishment of criteria for use of postoperative total parenteral nutrition, imaging, and invasive procedures. Prospective evaluation of all children treated for 20months on the new fully standardized protocol was conducted and compared to a retrospective cohort treated over 58months prior to standardization. Differences between outcomes before and after standardization were analyzed using regression analysis techniques to adjust for disease severity. RESULTS: Median follow-up time post discharge was 25 and 14days in the post- and prestandardization groups, respectively. Standardization significantly reduced postoperative abscess (9.8% vs. 17.4%, p=0.001) and hospital stay (p=0.002). Standardization reduced the odds of developing a postoperative abscess by four fold. CONCLUSION: Minimizing variability of care at all points in the treatment of perforated appendicitis significantly improves outcomes. TYPE OF STUDY: Prospective Cohort Study. LEVEL OF EVIDENCE: Level II.

Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue.

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Acetalated dextran encapsulated AR-12 as a host-directed therapy to control Salmonella infection.

AR-12 has been evaluated in clinical trials as an anti-cancer agent but also has demonstrated host-directed, broad-spectrum clearance of bacteria. We have previously shown that AR-12 has activity in vitro against Salmonella enterica serovar Typhimurium and Francisella species by inducing autophagy and other host immune pathways. AR-12 treatment of S. Typhimurium-infected mice resulted in a 10-fold reduction in bacterial load in the liver and spleen and an increased survival time. However, AR-12 treatment did not protect mice from death, likely due poor formulation. In the current study, AR-12 was encapsulated in a microparticulate carrier formulated from the novel degradable biopolymer acetalated dextran (Ace-DEX) and subsequently evaluated for its activity in human monocyte-derived macrophages (hMDMs). Our results show that hMDMs efficiently internalized Ace-DEX microparticles (MPs), and that encapsulation significantly reduced host cell cytotoxicity compared to unencapsulated AR-12. Efficient macrophage internalization of AR-12 loaded MPs (AR-12/MPs) was further demonstrated by autophagosome formation that was comparable to free AR-12 and resulted in enhanced clearance of intracellular Salmonella. Taken together, these studies provide support that Ace-DEX encapsulated AR-12 may be a promising new therapeutic agent to control intracellular bacterial pathogens of macrophages by targeting delivery and reducing drug toxicity.