Production of high-quality SARS-CoV-2 antigens for vaccine development and serological assays implementation

The development of novel and/or improved vaccines as well as the establishment of tools to monitor vaccine responses are two key factors to control COVID-19 pandemic, often requiring the manufacturing of significant amounts of high-quality SARS-CoV-2 antigens. In this work, we produced SARS-CoV-2 Spike (S) and the receptor binding domain (RBD) proteins in human or insect cell lines to be further used in (i) the implementation of serological assays for detection of antibodies against SARS-CoV-2 virus in the Portuguese population, and (ii) the development of a virosome-based COVID-19 vaccine candidate. Please click Download on the upper right corner to see the full abstract

Scalable process for high-yield production of PfCyRPA using insect cells for inclusion in a Malaria virosome-based vaccine candidate

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels

A novel inactivated virus system (InViS) for a fast and inexpensive assessment of viral disintegration.

The COVID-19 pandemic has caused considerable interest worldwide in antiviral surfaces, and there has been a dramatic increase in the research and development of innovative material systems to reduce virus transmission in the past few years. The International Organization for Standardization (ISO) norms 18,184 and 21,702 are two standard methods to characterize the antiviral properties of porous and non-porous surfaces. However, during the last years of the pandemic, a need for faster and inexpensive characterization of antiviral material was identified. Therefore, a complementary method based on an Inactivated Virus System (InViS) was developed to facilitate the early-stage development of antiviral technologies and quality surveillance of the production of antiviral materials safely and efficiently. The InViS is loaded with a self-quenched fluorescent dye that produces a measurable increase in fluorescence when the viral envelope disintegrates. In the present work, the sensitivity of InViS to viral disintegration by known antiviral agents is demonstrated and its potential to characterize novel materials and surfaces is explored. Finally, the InViS is used to determine the fate of viral particles within facemasks layers, rendering it an interesting tool to support the development of antiviral surface systems for technical and medical applications

Added value of combined acromiohumeral distance and critical shoulder angle measurements on conventional radiographs for the prediction of rotator cuff pathology

Purpose: To investigate the role of acromiohumeral distance (AHD) and critical shoulder angle (CSA) measurements from conventional radiographs (CR) in isolation and combined (prognostic index PIAHD-CSA) as predictors of full thickness rotator cuff tendon tears (RCT) and critical fatty degeneration (CFD; i.e. as much fat as muscle). Method: In this retrospective study AHD and CSA were measured in 127 CR. MR arthrograms served as reference standard and were screened for RCT and CFD. Statistical analysis for inter-reader agreement, Spearman's rank correlation, linear stepwise regression and logistic regression for AHD and CSA with ROC analyses including PIAHD-CSA were performed. Results: In 90 subjects (17 females, mean age 36.1 ± 14.1) no RCT were found on MR imaging and served as control group. In 37 patients (13 females, mean age 58.7 ± 13.2) ≥ one RCT was found. Inter-reader agreements rated between к = 0.42-0.82 for categorical and 0.91-0.96 for continuous variables. No significant correlation of AHD and CSA with either age or sex was seen (p = 0.28 and p = 0.74, respectively). Case group had significantly smaller mean AHD (8.7 ± 3.2 vs. 10.8 ± 2.2 mm; p < 0.001) and larger mean CSA (36.5 ± 4.5° vs. 33.1 ± 4.0°; p < 0.001). PIAHD-CSA increased diagnostic performance for prediction of RCT and CFD (AUC = 0.78 and 0.71), compared to isolated AHD (0.74 and 0.71) and CSA (0.71 and 0.66). Conclusions: AHD and CSA do not depend on age or sex but differ significantly between healthy and pathologic rotator cuffs. A decreased AHD is most influenced by infraspinatus muscle atrophy and fatty degeneration. Combined PIAHD-CSA increases diagnostic performance for predicting RCT and CFD. Keywords: Acromiohumeral distance; Conventional radiography; Critical shoulder angle; Fatty degeneration; Magnetic resonance arthrography; Magnetic resonance imaging; Rotator cuff tear

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels

Role of C-reactive protein in the bone marrow of Modic type 1 changes

Modic type 1 changes (MC1) are vertebral bone marrow lesions and associate with low back pain. Increased serum C-reactive protein (CRP) has inconsistently been associated with MC1. We aimed to provide evidence for a role of CRP in the tissue pathophysiology of MC1 bone marrow. From thirteen MC1 patients undergoing spinal fusion at MC1 levels, vertebral bone marrow aspirates from MC1 and intra-patient control bone marrow were taken. Bone marrow CRP, IL-1, and IL-6 were measured with enzyme-linked immunosorbent assays; lactate dehydrogenase (LDH) was measured with a colorimetric assay. CRP, IL-1, and IL-6 were compared between MC1 and control bone marrow. Bone marrow CRP was correlated with blood CRP and with bone marrow IL-1, IL-6, and LDH. CRP expression by marrow cells was measured with PCR. Increased CRP in MC1 bone marrow (mean difference: +0.22 mg CRP/g protein, 95% CI [-0.04, 0.47], p=0.088) correlated with blood CRP (r=0.69, p=0.018), with bone marrow IL-1β (ρ=0.52, p=0.029) and IL-6 (ρ=0.51, p=0.031). Marrow cells did not express CRP. Increased LDH in MC1 bone marrow (143.1%, 95% CI [110.7%, 175.4%], p=0.014) indicated necrosis. A blood CRP threshold of 3.2 mg/L detected with 100% accuracy increased CRP in MC1 bone marrow. In conclusion, the association of CRP with inflammatory and necrotic changes in MC1 bone marrow provides evidence for a pathophysiological role of CRP in MC1 bone marrow. This article is protected by copyright. All rights reserved

Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate.

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