Exploring insect cells versatility for production of influenza virus-like particles

A potential strategy to produce safer and broadly protective influenza vaccines is to co-express, in the same cell host, multiple hemagglutinins (HA) with a matrix protein (M1) which self-assemble in virus-like particles (VLPs). This study demonstrates the suitability of combining stable expression and the baculovirus-expression vector system (BEVs) in insect Hi5 cells for production of such multi-HA Influenza VLPs. Stable pools of Hi5 cells expressing two HAs were generated and later infected with a M1-encoding baculovirus at two cell concentrations (CCIs; 2×106 cells/mL and 3×106 cells/mL). The HA concentration in culture supernatant was followed over time, with more productive infections observed at higher CCIs. To extend the culture time, a re-feed strategy was implemented based on the identification of key nutrients which were exhausted during cell growth. Afterwards, supplemented cultures infected at a CCI of 4×106 cells/mL allowed a 4-fold increase in HA concentration, at harvest, when compared to cultures infected at a CCI of 2×106 cells/mL. The production of multi-HA influenza VLPs using the aforementioned strategy could be successfully scaled-up to 2L bioreactor cultures with even higher volumetric (1.5-fold) HA yields. To surpass the unpredictability of gene expression promoted by the random integration strategy mentioned above, the recombinase-mediated cassette exchange (RMCE) technology was explored. The feasibility of having two cassettes flanked by distinct pairs of flippase recognition target sites (FRTs) was evaluated. Unfortunately, significant cross-interaction was observed between the selected pairs. To circumvent this bottleneck, a backup strategy consisting in the co-expression of two genes from the same locus after implementation of one cassette system, in insect Sf9 cells, was attempted. However, the isolated clones showed low expression of both M1 and HA proteins. Ongoing work focuses on the isolation of clones tagged in high expression loci by fluorescence activated cell sorter technology. This work demonstrates how the versatility of insect cell expression technology can be explored to produce Influenza VLPs as vaccine candidates.A co-expressão de várias hemaglutininas (HA) e proteína da matriz (M1), no mesmo hospedeiro, formando partículas semelhantes a vírus (VLPs), constitui uma importante estratégia para desenvolver vacinas contra o vírus da gripe. Este trabalho mostra a combinação de uma linha celular estável de células de insecto com o sistema de expressão mediada por baculovírus para a produção deste tipo de VLPs. Foram estabelecidas duas populações de células de insecto Hi5, expressando duas HAs, posteriormente infectadas com um baculovírus contendo a proteína M1, a duas concentrações celulares diferentes (CCI; 2 e 3×106 cells/mL) sendo que a mais elevada demostrou ser a mais produtiva. De seguida, implementou-se uma estratégia baseada na adição de nutrientes específicos para prolongar o tempo de cultura. As culturas previamente suplementadas e infectadas a uma CCI de 4×106 células/mL produziram 4x mais HA comparativamente às culturas infectadas a uma CCI de 2×106 células/mL, não suplementadas. Esta estratégia foi também aplicada num biorreactor de 2L permitindo 1,5x mais produção, volumétrica, de HA comparativamente a experiências em pequena escala. De forma a ultrapassar a imprevisibilidade de uma integração aleatória, foi explorado o sistema de troca de cassete mediado por recombinase (RMCE). A viabilidade de um sistema com duas cassetes integradas flanqueadas por diferentes locais de reconhecimento (FRTs) foi avaliada, tendo sido observada a interação entre ambos os pares selecionados. Como segunda estratégia, foi implementado um sistema com uma cassete para co-expressão de dois genes em simultâneo, em células de insecto Sf9. Porém, os clones isolados mostram fraca expressão de M1 e HA, pelo que uma estratégia de isolamento de clones com expressão génica mais forte está em desenvolvimento utilizando uma tecnologia de sorteamento. Assim, este trabalho demonstra a versatilidade da tecnologia aplicada em células de insecto, que pode ser explorada para produzir VLPs multivalentes, com potencial para se tornar a próxima geração de vacinas para o vírus da gripe

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research