Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Development of a personalized dendritic cell vaccine and single-cell RNA sequencing-guided assessment of its cell type composition.

BACKGROUND AIMS: Dendritic cell (DC)-based immunotherapy is a promising approach to treat cancer; however, there is no consensus on the manufacturing processes. Cell type heterogeneity in products manufactured by various methods is understudied and may elicit safety concerns from the regulatory perspective. METHODS: We characterized the cell type composition of a recently developed DC vaccine, CUD-002, consisting of DCs loaded with mRNA encoding personalized tumor neoantigens (NCT05270720). RESULTS: Using single-cell transcriptomic analysis as an unbiased approach, we found that >80% cells in the final product were DCs and the rest primarily comprised myelocytes and lymphocytes. Subsequent fluorescence-activated cell sorting analyses confirmed these cellular identities. These results indicate that unintended cells originate from leukapheresis, the first step of the manufacturing process, and thus likely safe. Consistently, no overt toxicity or tumorigenicity was observed in mice inoculated with CUD-002. CONCLUSIONS: Considering that leukapheresis is a widely used procedure for collecting diverse peripheral blood cell types to manufacture various cytotherapies, this study establishes a workflow to analyze and address regulatory considerations on cell type heterogeneity.

Current advances in the algae-made biopharmaceuticals field.

INTRODUCTION: The biopharmaceuticals industry demands new production platforms to address several challenges; such as cost reduction to make biologics accessible in low-income countries, safety enhancement of the product, development of products administered by noninvasive routes, and expansion of potential biosimilars and biobetters. Microalgae are emerging hosts for biopharmaceuticals production with the potential to meet such requirements. AREAS COVERED: Nowadays successful cases on the production of vaccines, antibodies, antimicrobial peptides, growth factors/cytokines, and hormones in algae have been reported. This review comprises an updated outlook covering protein expression strategies, a compilation of functional biopharmaceuticals produced in algae, and companies investing in this technology. EXPERT OPINION: Key perspectives for the field include optimizing yields, scaling up production and completing preclinical trials. The experience from the field of plant-made biopharmaceuticals is commented as a key reference that will aid in the development of the algae-made biopharmaceuticals field.

Innovative Approaches to Improve Anti-Infective Vaccine Efficacy.

Safe and efficacious vaccines are arguably the most successful medical interventions of all time. Yet the ongoing discovery of new pathogens, along with emergence of antibiotic-resistant pathogens and a burgeoning population at risk of such infections, imposes unprecedented public health challenges. To meet these challenges, innovative strategies to discover and develop new or improved anti-infective vaccines are necessary. These approaches must intersect the most meaningful insights into protective immunity and advanced technologies with capabilities to deliver immunogens for optimal immune protection. This goal is considered through several recent advances in host-pathogen relationships, conceptual strides in vaccinology, and emerging technologies. Given a clear and growing risk of pandemic disease should the threat of infection go unmet, developing vaccines that optimize protective immunity against high-priority and antibiotic-resistant pathogens represents an urgent and unifying imperative.