RNA Vaccines: Yeast as a Novel Antigen Vehicle

Abstract

In the last decades, technological advances for RNA manipulation enabled and expanded its application in vaccine development. This approach comprises synthetic single-stranded mRNA molecules that direct the translation of the antigen responsible for activating the desired immune response. The success of RNA vaccines depends on the delivery vehicle. Among the systems, yeasts emerge as a new approach, already employed to deliver protein antigens, with efficacy demonstrated through preclinical and clinical trials. β-glucans and mannans in their walls are responsible for the adjuvant property of this system. Yeast β-glucan capsules, microparticles, and nanoparticles can modulate immune responses and have a high capacity to carry nucleic acids, with bioavailability upon oral immunization and targeting to receptors present in antigen-presenting cells (APCs). In addition, yeasts are suitable vehicles for the protection and specific delivery of therapeutic vaccines based on RNAi. Compared to protein antigens, the use of yeast for DNA or RNA vaccine delivery is less established and has fewer studies, most of them in the preclinical phase. Here, we present an overview of the attributes of yeast or its derivatives for the delivery of RNA-based vaccines, discussing the current challenges and prospects of this promising strategy.

Keywords: antigenic delivery; delivery vehicle; mRNA vaccines; therapeutic vaccines; whole yeast vaccine.

Figure 1

A general summary of composition, features, and applications of mRNA vaccines. The cap (5′) and a poly-A tail (3′) flanking the ORF are required for the translation of target proteins and represent the typical features present in the structure of mRNA vaccines. These vaccines, however, have drawbacks such as low stability, inherent toxicity, and poor distribution in the body. Codon optimization, chemical changes, and the usage of carrier molecules are being employed to solve these issues. For medicinal applications, mRNA vaccines have demonstrated promise in treating infectious, neoplastic, and autoimmune illnesses, opening the path for clinical improvements.

Figure 2

(A) Heat treatment promotes an increase in the exposure of β-1,3-glucans on the yeast surface (heating at 60 °C for at least 1 h), enhancing the adjuvant capacity of the yeast, favoring its recognition and binding to receptors present in antigen-presenting cells. (B) Dendritic cells recognize yeast β-1,3-glucans mainly through the Dectin-1 receptor that promotes and facilitates phagocytosis (1). After the uptake, the phagosome is formed, where the yeasts are processed. The fragmented antigens can be presented by MHC-I (cytosolic proteolysis) leading to the activation of cytotoxic TCD8+ lymphocytes, or by MHC-II (endolysosomal proteolysis) inducing the activation of TCD4+ Helper lymphocytes. (2) Inflammatory cytokines such as TNF-α, IL-6, IL-8, and IL-1β or IL-12, IL-23, and IL-27, released by activated dendritic cells polarize T cells to Th1 and Th17 profiles (3).

Figure 3

Main components of expression vectors used to carry mRNA vaccines by yeast. The choice of the promoter, constitutive or inducible, may influence the successful transcription of the target gene (also depending on the phagocytic cell involved, macrophage, or dendritic cell). The IRES sequence (derived from EMCV) prevents the translation of vaccine antigens from occurring within yeast cells before phagocytosis. In addition to these components, the poly(A) sequence is important for stabilizing mRNA. It is necessary to add a marker, preferably by auxotrophic, to select recombinant yeasts.

Figure 4

Yeast-based vaccine modalities. (A) Yeast, as a complete organism, can carry plasmids with the desired antigen gene inside them. The immunostimulatory effects result from the interaction between the yeast cell wall components and macrophage and dendritic cell receptors. (B) β-glucan microcapsules or nanocapsules are particles obtained after physical-chemical treatment to remove the mannoprotein layer. Differences in size between the whole cell and the β-glucan particles can influence the phagocytosis process and the delivery and dissemination of antigens through lymphatic vessels.

Figure 5

Main elements present in expression vectors for RNAi delivery. (A) Cassette with a promoter recognized by mammalian cells. The cassette should also contain a leader sequence just after the promoter, and it is recommended to flank the target gene shRNA by endogenous miRNA (e.g., miR30). Uracil is the selection marker usually employed. (B) Cassette for expression with yeast promoters (constitutive or inducible). URA3/TRP1 sequences are loci for cassette integration and auxotrophic marker genes.