== A

== A. RBDs. Inside a murine model, we proven how the yeast-produced RBD-dimer induces protecting and powerful antibody reactions, which would work to enhance immunization. This scholarly research created the candida program for creating SARS-CoV-2 RBD-dimer immunogens, offering a guaranteeing system and pipeline for future years constant upgrading and creation of SARS-CoV-2 vaccines. == Author summary == SARS-CoV-2 continually circulates, resulting in infections and re-infections globally. COVID-19 vaccines can provide safety against disease and death caused by SARS-CoV-2. However, the immune reactions induced by vaccination will wane gradually. In addition, fresh SARS-CoV-2 sub-variants emerge and display immune evasion. Therefore, inoculating with the updated COVID-19 vaccines is recommended for enhancing safety against the circulating SARS-CoV-2 sub-variants.Pichia pastorisis an JQEZ5 eukaryotic system that enables fast and low-cost producing proteins with easy scale-up. In this study, we developed a strategy for generating SARS-CoV-2 RBD-dimer immunogens in glycoengineeredPichia pastoriswith homogeneous glycosylation and high stability. JQEZ5 The RBD antigen can be updated with the sequence of circulating sub-variants. In addition, mouse experiments shown the yeast-produced RBD-dimer vaccines induced powerful and protecting immune reactions. This study provides a simple and promising approach to produce yeast-derived SARS-CoV-2 RBD-dimers like a improving vaccine and guides the basis for the production of the additional vaccines inPichia pastoris. == Intro == Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a severe danger to global health, having caused almost 7 million deaths to day (https://covid19.who.int/). To combat the coronavirus disease 2019 (COVID-19) kanadaptin pandemic, vaccines have been developed using numerous platforms [13], JQEZ5 including recombinant protein subunit vaccines[1], inactivated viruses vaccines[4], mRNA vaccines [5], DNA vaccines, and virus-vectored vaccines [6,7]. However, SARS-CoV-2 variants continually emerge and circulate, escaping herd immunity. Additionally, the immune reactions induced by vaccination and breakthrough illness will gradually wane [8,9]. Consequently, the administration of booster vaccines is vital to enhance safety against the circulating SARS-CoV-2 variants, especially for the elderly, immunocompromised individuals, and people suffering from chronic diseases such as diabetes and hypertension. SARS-CoV-2 spike (S) protein is responsible for recognizing the sponsor cellular receptor ACE2 through its receptor-binding website (RBD) [10]. Therefore, the RBD is an attractive target to elicit neutralizing antibodies. Previously, we shown that tandem-repeat RBD-dimers induced higher neutralizing antibody titers than traditional monomeric RBDs and developed the COVID-19 protein subunit vaccine ZF2001 [1,11]. Furthermore, we designed a heterologous RBD-dimer strategy to JQEZ5 develop bivalent vaccines JQEZ5 eliciting broad-spectrum immune responses [12]. RBD-dimer immunogens have also been used in mRNA, DNA and adenovirus-vector centered vaccines to induce powerful immune responses [1316]. In the future, high-efficiency vaccines will likely be given regularly like the current influenza vaccines. Therefore, the COVID-19 vaccines should be safe, effective, easy to upgrade, amenable to mass production, and low cost for use globally. COVID-19 protein subunit vaccines have been successfully developed based on numerous immunogens [1,17,18], adjuvants [17,1922] and manifestation systems [11,18,22]. Candida manifestation systems, likePichia pastoris[23], present simple and cost-effective vaccine developing and distribution platforms. They are anticipated to enhance global vaccine convenience and equity, particularly for low- and middle-income countries (LMICs). Recent advancements have been made in expressing SARS-CoV-2 RBD antigens usingP.pastoris[2433]. In contrast to mammalian and insect cells, recombinant proteins secreted from the candida cells are revised by hyperglycosylation at N-glycosylation sites. The published studies also statement varying examples of glycosylation of RBD protein produced byP. pastorisdue to the N-glycosylation sites at N331 and N343 [2931,34]. Hyperglycosylation can obscure immune epitopes and impact protective immune responses [35]. In addition, the lack of protein uniformity caused by hyperglycosylation complicates protein purification processes and potentially affects production consistency, therefore raising issues about the quality of large-scale production. To obtain the glycosylated RBD protein with better uniformity, experts have attempted to mutate the N-glycosylation sites or use glycoengineered candida as an expression sponsor [24,27,32]. Among these studies, Corbevax [36] and Abdala [37] are based on the RBD monomer antigen produced inP.pastorisand have been demonstrated to be.