Towards the future exploration of mucosal mRNA vaccines against emerging viral diseases; lessons from existing next-generation mucosal vaccine strategies

Abstract

The mRNA vaccine platform has offered the greatest potential in fighting the COVID-19 pandemic owing to rapid development, effectiveness, and scalability to meet the global demand. There are many other mRNA vaccines currently being developed against different emerging viral diseases. As with the current COVID-19 vaccines, these mRNA-based vaccine candidates are being developed for parenteral administration via injections. However, most of the emerging viruses colonize the mucosal surfaces prior to systemic infection making it very crucial to target mucosal immunity. Although parenterally administered vaccines would induce a robust systemic immunity, they often provoke a weak mucosal immunity which may not be effective in preventing mucosal infection. In contrast, mucosal administration potentially offers the dual benefit of inducing potent mucosal and systemic immunity which would be more effective in offering protection against mucosal viral infection. There are however many challenges posed by the mucosal environment which impede successful mucosal vaccination. The development of an effective delivery system remains a major challenge to the successful exploitation of mucosal mRNA vaccination. Nonetheless, a number of delivery vehicles have been experimentally harnessed with different degrees of success in the mucosal delivery of mRNA vaccines. In this review, we provide a comprehensive overview of mRNA vaccines and summarise their application in the fight against emerging viral diseases with particular emphasis on COVID-19 mRNA platforms. Furthermore, we discuss the prospects and challenges of mucosal administration of mRNA-based vaccines, and we explore the existing experimental studies on mucosal mRNA vaccine delivery.

Fig. 1. MALT and mucosal immune response.

The MALT can be functionally divided into 2 portions, the inductive and the effector sites. The organized lymphoid tissue composed of lymphoid follicles, present along the GIT (GALT) and the respiratory tract (NALT) represent the inductive sites where immune response is initiated. Overlying the follicles are specialized epithelium which in the Peyer’s patches is called the follicle-associated epithelium (FAE). This overlying epithelium is equipped with functionally active microfold cells (M cells) which are involved in antigen sampling from the lumen and delivers these luminal antigens to the underlying DCs and macrophages (APCs) in the subepithelial follicles via transcytosis. Some of the underlying DCs and Macrophages also directly sample antigens from the lumen by the extension of transepithelial dendrites across the epithelium or by occasional migration into the lumen. Following antigen capture, the APCs delivers and present the antigens to the T cells and B cells present in the follicles to induce an antigen-specific immune response. The activated T and B cells then exit the submucosa via the lymphatics to the mesenteric lymph nodes where the immune response may be further exaggerated before finally draining into the systemic circulation. These activated cells then express mucosal homing receptors such as CCR9 and CCR10 and are guarded by gradient of chemokines such as CCL25 and CCL28 present in the mucosa to finally exit the blood, a process mediated by integrins and adhesion molecule α4β7 and MAdCAM-1 respectively. At the effector site where the effector functions are carried out, activated T cells go on to become effector cells and/or tissue-resident memory cells. Activated B cells undergo class-switch to become IgA+ B cells and plasma cells which add joining chains to secrete polymeric IgA. These polymeric IgA are transported transcellular to the lumen following binding to polymeric Ig receptor (pIgR) as secretory IgA (sIgA) which lines the mucus and functions in trapping microbes.

Fig. 2. IVT-mRNA transcription and purification.

Crude IVT-mRNA which has been generated synthetically from a linearized DNA template using polymerases (T3/T7) often contains a mixture of the mRNA molecules with different categories of impurities. The crude mRNA is initially subjected to extraction and precipitation processes which remove some but not all the impurities, final HPLC or FPLC treatment generates pure grade mRNA molecules.

Fig. 3. Immune response to mRNA vaccines.

a Innate Immune response to mRNA vaccines The delivery of the mRNA molecules into the cytosol is followed by the detection of the mRNA molecules by TLRs. Double-stranded RNA molecules co-delivered as impurities or produced from SAM or as secondary structures are additionally detected by RLRs (RIG-1, MDA-5). These drive cytokine and chemokine responses that recruit more innate cells to the injection site. Amplified interferon response can result in the activation of OAS/PKR whose signals impede translation, protein expression and antigen presentation. b Adaptive Immune response to mRNA vaccines. Following In vivo delivery of the mRNA vaccine, the mRNA molecules with the delivery vehicles are (1) uptaken by cells such as DCs at the site of injection by endocytosis with subsequent delivery into the endosomes. This is followed by (2) endosomal escape of the mRNA molecules into the cytosol and subsequent (3) translation in the cytosol to produce the encoded protein. The produced protein may be retained in the cytosol where it is subsequently channelled for (4,5,6) proteasomal-MHCI pathway which would eventually drive a CD8 T cell response. Some of the proteins may also become (7) membrane-bound and expressed on the surface or (8) secreted/shed. Some of the secreted or expressed proteins may be (9) recycled by endocytosis and subsequently channelled through the MHCII-restricted presentation to eventually drive CD4 T cell response. B-cell and T-cell responses occur by virtue of their interactions with the secreted or membrane-bound proteins and MHC-antigen complexes respectively. (10) These adaptive responses prevent infection and facilitate the elimination of the pathogen upon encounter through antibody production, cytokine release and cytotoxic activities.

Fig. 4. Summary of mRNA delivery systems.

This figure provides an overview of the existing in vivo delivery systems and the components of the various classes.

Fig. 5. Comparison between mucosal and invasive mRNA vaccination.

The immunological benefits of mucosal and invasive vaccine administration as proposed for mRNA vaccines. (1) Following mucosal delivery, the vaccine uptake induces responses at inductive sites in the (2) mucosal lymphoid tissue from where antigen-specific lymphocytes are (3) transported systemically in the blood and home to the primary and distant mucosal surfaces. (4) This results in the production sIgA and the presence of the antigen-specific B and T-lymphocytes at different mucosal sites and eventually preventing (5) mucosal colonization or disease transmission with abundant IgG in the blood to prevent or limit viraemia. In contrast, (6) invasive routes of delivery induce a very effective systemic response with abundant IgG present to prevent viraemia, but (7) this induces a relatively weak mucosal response with little to no sIgA, which may not effectively prevent mucosal infection or transmission.

Fig. 6. Challenges encountered in the mucosal environment.

Following sublingual immunization, the antigen faces challenges associated with the (1) crossing of the stratified epithelium and (2) salivary dilution of antigen. With oral immunization, the vaccine faces challenges in different regions along the GIT. In the stomach, there are challenges due to the (1) destructive action of the acidic environment and (2) the degradative action of enzymes. In the small intestine, the challenges faced are those impacted by the (3) pH variation, (4) degradative enzymes and (5) intestinal mucus in addition to the intrinsic immunotolerance. At the rectal mucosa, the problem faced is those impacted by the (5) thick mucus layer and the tolerogenic microenvironment. Following nasal immunization, at the respiratory mucosa, the vaccine faces challenges due to the (1,2) mucociliary action of the ciliated epithelium which continually pushes the antigen, a process that reduces residence time, the (2) low-grade enzymatic action of RNAses and also the intrinsic immunotolerance. At the vaginal mucosa, the challenges are due to the mucus layer which thickens with increased estradiol (E2) level and reduces uptake as well as the immunotolerance with the low immunogenic response which is often limited to the local genital tract. The epithelial barrier formed by the epithelial layer of cells is a limiting factor to vaccine uptake which is common in all mucosal compartments.