No abstract

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

Objective: to evaluate the effectiveness of the telephone intervention for promoting self-care related to physical activity and following a diet plan in users with diabetes, compared to conventional monitoring of users over a six-month period. Method: this was a randomized clinical trial, which included 210 users with diabetes, linked to eight Primary Health Units of Belo Horizonte, Minas Gerais. The experimental group (104 members) received six telephone interventions over the six-month monitoring; the control group (106 members) received conventional monitoring. To evaluate the self-care practices related to physical activity and following a healthy eating plan, in both groups, the self-care questionnaire was applied before the intervention and at three and six months after its start. Results: the mean effect of self-care scores in the experimental group was 1.03 to 1.78 higher than the control group, with progressive and significant improvement (p<0.001). Conclusion: the results indicate that the telephone intervention had a beneficial effect on diabetes self-care. The primary identifier of the clinical trials registry was: RBR-8wx7qb.

Conformationally complex membrane proteins (MPs) are therapeutic targets in many diseases, but drug discovery has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of virus-like particles (VLPs), preserving their native lipidic environment. Here, we implemented a site-specific recombinase-mediated cassette exchange (RMCE) strategy to establish a reusable HIV-1 Gag-expressing insect cell line for fast production of target MPs on the surface of Gag-VLPs. The Sf9 cell line was initially tagged with a Gag-GFP-expressing cassette incorporating two flipase recognition target sites (FRTs), one within the fusion linker of Gag-GFP. The GFP cassette was afterwards replaced by a Cherry cassette via flipase (Flp) recombination. The fusion of Gag to fluorescent proteins enabled high-throughput screening of cells with higher Gag expression and Flp-mediated cassette exchange ability, while keeping the functionality of the VLP scaffold unaltered. The best cell clone was then Flp-recombinated to produce Gag-VLPs decorated with a human β2-adrenergic receptor (β2AR). Release of a fluorescently labeled β2AR into the culture supernatant was confirmed by immunoblotting, and its co-localization with Gag-VLPs was visualized by confocal microscopy. Furthermore, the differential avidity of β2AR-dsplaying Gag-VLPs versus "naked" Gag-VLPs to an anti-β2AR antibody measured by ELISA corroborated the presence of β2AR at the surface of the Gag-VLPs. In conclusion, this novel insect cell line represents a valuable platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.

Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells.Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 10 6 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

Objective: To develop, validate the content and conduct the cultural adaptation of the Compasso protocol for promoting adherence to self-care practices in diabetes via telephone intervention. Methods: Methodological research for tool development, following three stages: protocol development; content validation; and cultural adaptation. Fourteen professionals affiliated to the fields of healthcare, applied linguistics and statistics participated in the stages of development and content validation, while 341 individuals with diabetes mellitus took part in the adaptation stage. Data for validation and adaptation were collected through the web platform e-Surv and analyzed in the R environment. Results: The Compasso protocol includes the following domains: behavioral and psychosocial aspects, support network, barriers, and self-care practices. The assessment of the protocol presented good agreement between the evaluators, with a mean CVI 0.96. Conclusion: The content of the Compasso protocol was considered to be validated and culturally adequate to promote adherence to the practices of diabetes self-care via telephone intervention. ResumoObjetivo: Realizar a construção, validação de conteúdo e adequação cultural do protocolo Compasso para promover a adesão às práticas de autocuidado em diabetes via intervenção telefônica. Métodos: Pesquisa metodológica de elaboração de instrumentos abrangendo as etapas: construção do protocolo; validação de conteúdo; adequação cultural. Participaram das fases de construção e validação um total de 14 profissionais das áreas da Saúde, Linguística e Estatística, e, da fase de adequação, 341 usuários com diabetes mellitus. Os dados para validação e adequação foram coletados através da plataforma web e-Surv e analisados no ambiente R. Resultados: O protocolo Compasso aborda os seguintes domínios: aspectos comportamental e psicossocial, rede de apoio, barreiras e práticas do autocuidado. A avaliação do protocolo apresentou boa concordância entre os avaliadores, com IVC médio de 0,96. Conclusão: Considera-se validado o conteúdo do protocolo Compasso e adequado culturalmente para promover a adesão às práticas de autocuidado em diabetes via intervenção telefônica.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at −80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate.

We report a 13-year-old boy with ectodermal dysplasia, ectrodactyly, and syndactyly, hypospadias, photophobia, conductive hearing loss, and perioral papillomatosis. His father had ectrodactyly and hypotrichosis. The clinical picture suggested ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome. The presence of perioral papillomatosis, classically seen in Goltz syndrome, has been reported only once before in EEC syndrome.