www.nature.com/npjvaccines REVIEW ARTICLE OPEN Towards the future exploration of mucosal mRNA vaccines against emerging viral diseases; lessons from existing next-generation mucosal vaccine strategies 1✉ 2 Sodiq A. Hameed , Stephane Paul , Giann Kerwin Y. Dellosa1, Dolores Jaraquemada3 and Muhammad Bashir Bello4 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 1234567890():,; 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. npj Vaccines (2022)7:71 ; https://doi.org/10.1038/s41541-022-00485-x INTRODUCTION strategy induces a robust systemic immune response, there is Since time immemorial, emerging viral diseases have been threats often a weak local mucosal immunity which may be inefficacious to humanity all over the world1. They are responsible for the death in curbing mucosal infection. In contrast, the mucosal route of of millions of people annually and constitute a significant problem vaccination not only induces a very strong local immunity but also to the healthcare system, social stability, and food security, provokes a robust protective systemic response, hence, forming especially in resource-poor countries. Although the major drivers an effective strategy to prevent mucosal colonization5,6. for the emergence and spread of these deadly diseases are Over the last 3 decades, epidemics of emerging viral diseases urbanisation, climate change and modernisation of the transport including Avian influenza, Ebola, Severe acute respiratory syn- drome (SARS), Middle East respiratory syndrome (MERS) and industry; poor sanitation and limited access to health services also several others have occurred in different parts of the world. The contribute substantially to the burden of these diseases2. most recent global health threat is Coronavirus disease 19 Emerging viral diseases are generally categorised into newly (COVID-19) caused by the novel corona virus called SARS-COV-2, emerged viral diseases that have not been known to previously be which emerged in Wuhan city of China in December 20197. Within associated with any disease in man, and re-emerging viral diseases a few months of its emergence, COVID-19 had spread to all that were recognized previously but have adapted to become continents on earth, affecting over 265 million people with more major health threats in a population3. Unsurprisingly, most than 5.2 million deaths as of 6th December 20218. Indeed emerging viral diseases are caused by RNA viruses which are COVID-19 has proven to be a global public health emergency that generally more prone to mutation than their DNA counterparts. has the potential to ravage the economy and social stability of The high mutation rate in those viruses, largely driven by genetic several countries. So far, the disease has placed millions of people recombination events and poor proofreading ability of the viral at the risk of falling into extreme poverty, while the number of replicases, is believed to be responsible for the rapid evolution undernourished people has been projected to be more than 800 and continued emergence of new often deadly and difficult to million before the beginning of the year 20219. Although most control infectious pathogens that quickly adapt to escape vaccine- countries took measures such as border closures, trade restrictions induced population immunity4. and total lock down to curtail the spread of the disease, the The mucosal route remains the portal of entry for most of these actions dramatically disrupted domestic and international food pathogens from where they disseminate further to cause a supply chains, aggravating the already existing food insecurity, systemic disease. However, most of the vaccines available against especially in resource-poor nations. Given this huge socio- these pathogens are administered systemically. Although this economic impact of COVID-19 and indeed all other emerging 1 Univ Lyon, Université Claude Bernard Lyon 1, 69100 Villeurbanne, France. 2CIRI – Centre International de Recherche en Infectiologie, Team GIMAP, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR530, CIC 1408 Vaccinology, F42023 Saint-Etienne, France. 3Universidad Autónoma de Barcelona, 08193 Cerdanyola, Spain. 4 Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Usmanu Danfodiyo University PMB, 2346 Sokoto, Nigeria. ✉email: Hameedsodiq71@gmail.com Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 2 1234567890():,; 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. viral diseases particularly on the most marginalized populations, in the nucleus15. More so, when intracellularly processed and there is a need to develop effective countermeasures to reduce presented by antigen-presenting cells, the products of mRNA the burden of emerging viral diseases (Figs. 1–6). vaccines are able to stimulate robust cell mediated and humoral To date, vaccines are considered among the most successful immune responses in the immunised hosts16. mRNA molecules tools for public health interventions against emerging viral are the templates for protein synthesis in the cytoplasm of a cell. pathogens10. They have historically been very effective in They are however highly unstable, a limitation that has delayed eradicating or at least reducing mortality and morbidity due to the progress in the application of mRNA technology for infectious several infectious diseases. Whereas most conventional vaccines disease control. However, with the discovery of strategies for have contributed immensely towards infectious disease control, enhancing the stability of mRNA17, a growing substantial number they are developed using strategies that are cumbersome, risky of literature now exists that highlights the role of mRNA in modern and time consuming, making them unsuitable under pandemic vaccinology. Finally, these mRNA-based vaccines are being situations where accelerated vaccine development is a prior- developed for parenteral administrations via the intramuscular, ity11,12. The recent advances in recombinant DNA technology have intradermal, subcutaneous and intravenous routes18. Although paved the way for the emergence of the state-of-the-art “next these routes have recorded a high degree of success in mRNA generation” vaccine technologies. Courtesy of bioinformatics and vaccinology, the comparative benefits of the mucosal delivery molecular biology, rationally designed effective vaccines based on route would be of high value in tackling the viruses at the portal synthetic peptides, replication-competent/deficient virus vectors, of entry. as well as nucleic acid (DNA and mRNA) have emerged13. Among these vaccine platforms, mRNA vaccines have many advantages that make them promising tools for combating emerging viral THE MUCOSAL-ASSOCIATED LYMPHOID TISSUE (MALT) diseases. First of all, they are simple to generate, safe to handle, Numerous studies have outlined the structural and immunological easily taken up and processed using the host’s translational architecture of the Mucosal Associated Lymphoid Tissue (MALT) in machinery14. They can also be chemically manufactured at man. However, for the purpose of this review, general salient industrial scales without the need for animal or cellular features are outlined while region-specific peculiarities which can components. Furthermore, they do not need to be trafficked into influence the uptake of and response to mRNA-based vaccines the nucleus for expression, making them safe from integration are discussed. Generally, the mucosal tissue forms the most into host genome and less susceptible to degradation by enzymes extensive physical barrier that delineates the internal organs from npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 3 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. the physical environment19–21. Hence structural and functional then home to mucosal sites guided by adhesion molecules on adaptations have evolved to maintain a delicate homeostatic endothelial surfaces such MAdCAM-1, to elicit effector function28. balance allowing a large degree of tolerance as well as being The diffuse areas of the lymphoid population present in the reactive towards an invading pathogen. Structurally, the mucosal subepithelial lamina propria have been described mostly as the tissue consists of an epithelial layer covered in mucus which effector sites of the MALT which houses homing activated cells or ranges from, simple cuboidal, columnal (simple or pseudostrati- in-situ activated cellular population (e.g., T-independent activated fied), the stratified squamous epithelium (with or without B cells)25. Generally, the MALT consists of bacterial protease- keratinization). These are linked together laterally by tight resistant IgA2-producing B-cell population, but this is highest in junctions with junctional proteins, and basally by a basement the large intestine25,29. An important adaptation of IgA formed membrane with associated germ cells or other cells of immuno- from the MALT is the secretory form (SIgA) which cross links large logical origin22,23. These are then underlaid with lamina propria of antigens with multiple epitopes in a neutralization reaction either loose connective tissue which often houses organized and loose in the lumen, intraepithelial or at the lamina propria. These SIgAs, aggregates of lymphoid cells and innate immune cells22,24. This formed from complexing of polymeric IgA to its receptor, are general architecture although regionally specialized, affords trapped in the mucus layer of the MALT which increases in the mucosal tissue its functional role of ensuring absorption of thickness and composition from the oral cavity to the large nutrients, preventing pathogen colonization by providing intestine. The mucus consists of two layers, a thinner inner layer sentinel functions as well as eliciting defence when the barrier is that is sterile and difficult to dislodge and an outer non-sterile compromised24. The Mucosal Associated Lymphoid tissues layer easy to dislodge27. Together both layers consist of mucin (MALT), structures found in the lamina propria, are composed of 5AC (MUC5AC) and MUC6, produced principally by goblet cells naive, activated and memory B cells and T cells as well as antigen and have a thickness ranging from ~15–400 μm30. The MALT is present cells (Dendritic cells and Macrophages). These are overlaid subdivided into different compartments depending on the with specialized epithelia structures called the Follicle-associated anatomical location. These are discussed in the following sections. epithelium containing functionally active microfold cells (M-cells) which facilitate transduction of antigens (sentinel functions) to the The gut-associated lymphoid tissue (GALT) follicles—inductive sites. In fact, the MALT has been described as The Peyer’s patches represent the main inductive sites of the home to over 80% of effector B-cell population in the body24,25. GALT31. They are roughly oval structures concentrated in the distal Other inductive sites include tonsils in the palate and adenoids in one-third of the ileum although extend into the jejunum with a the oropharynx among others25,26. Following antigenic stimula- few numbers in the duodenum identified. This makes the ileum a tion, class cell-switching and affinity maturation of B cells occur in desirable target for vaccine antigen uptake as both surface area the germinal centre of the follicles with the associated activated and propensity for uptake is maximized at this site31,32. Hence, a CD4+ T cells giving rise to effector cells which migrate to the vaccine must be designed to withstand the site-specific adapta- interfollicular subepithelial area27. The interfollicular areas are tions of the GI and become easily absorbed at this site. Studies composed of abundant T-cell population, innate lymphoid cells, have shown that Peyer’s patches appear as early as the 3rd along with macrophages, and DCs. An important mechanism of trimester of life and climaxing in number into the late third induction-effector distribution of activated cells along different decade after which their numbers decrease31. The development of regions of the MALT is influenced by the exiting of activated cells Peyer’s patches is also influenced by gut microbiome through diet into circulation through the high endothelia venules (HEVs) which and environmental influences, hence, making this site a desirable Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 4 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. target in infants and young adults. These organized lymphoid intestinal epithelia cells armed with vast array of extracellular and tissues of the GALT are composed of abundant number of intracellular sensors such as Toll-Like Receptors (TLRs)36. Indeed, lymphoid cells, most naive, ranging from B cells, T cells, microbiota interaction with receptors on the apical site of IEC macrophages and dendritic cells (DCs) including an overlying mostly triggers tolerogenic pathways orchestrated by TGF-β, BAFF FAE that contains abundant M cells32,33. In addition, double- and APRIL from enterocytes and intestinal DCs which drive IgA negative (CD4− CD8-) mucosal-associated Invariant T cells (MAIT) production in resident B cells27. However, stimulation from the with an invariant TCR which is restricted to an MHC-related 1 microbiota may trigger a potent inflammatory response when (MR1) molecule, have been reported abundant in the GALT. These there is a breach to the intestinal epithelial barrier due to damages cells are capable of producing different cytokines in response to leading to the release of cytokines such as IL-1β and IL-6, and microbial invasion and this is largely dependent on the transcrip- chemokines such as IL-8 (CXCL-8) and RANTES (CCL5). Further- tional factors implicated in the responses34. Constituent GALT DCs more, alarm signals can be largely triggered from the apical IECs plays a significant role in the induction-effector function of GI thereby breaking the tolerance threshold following the interaction mucosal. Studies have shown GALT-associated MHCII+ CD11c+ of microbial structures (such a Lipopolysaccharides component of CD11b+ DCs to be divided into two subsets; the CD103+ CX3CR1− the bacterial cell wall) with PRRs on the extracellular or which captures antigen to induce IgA production and imprints intracellular milieu of the GI epithelium37. immunoregulatory T-cell phenotype, and which is responsible for oral tolerance following the ingestion of protein antigen. The second subset is the CD103- CX3CR1+ DCs which includes the Nasopharynx associated lymphoid tissue (NALT) intestinal macrophages; these cells generally induce an inflam- The NALT has been described as the main inductive site of the matory response and are responsible for imprinting TH-17 respiratory mucosa, being the equivalent of Peyer’s patches in the phenotype, being implicated in colitis35. Additionally, it was gut in its structural organization. Generally, the NALT includes reported that aside the use of transepithelial dendrites extension the organized lymphoid structures in the nose, the bronchial and to capture antigen from the intestinal lumen for indirect antigen the larynx. This also encompasses the oropharyngeal lymphoid sampling, the intestinal CX3CR1+ macrophages transiently structures such as the palatine tonsils and the adenoids which migrate into the intestinal lumen to capture antigens directly, to altogether form the Waldeyer’s ring. The organized lymphoid trigger cascades of downstream immune response signalling36. structures houses immune cells for inducing T and B cell Sentinel functions in the GI is also carried out extensively by the responses38. All the aforementioned lymphoid structures of the npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 5 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. respiratory system develop first in early life and persist beyond lymphoid structures33,42. The airway is lined by specialized childhood39. This phenomenon can possibly be attributed to the epithelium which functions in response against microbial invasion. induction of antigens in breathed air passing through the nares The epithelial cells are equipped with PRRs which can sense before getting to the lungs. Therefore, in a similar vein, microbial ligands to trigger the release of cytokine and vaccination strategies targeting the NALT in the nasal epithelium chemokines that stimulate immune cells in the NALT. Additionally, at the precise particle size could stimulate uptake induction and airway epithelia cells have been reported to produce BAFF (B Cell- elicit protection against a respiratory pathogen that opportunis- activating factor of the TNF family) and APRIL (A Proliferation tically wants to colonize the nasal mucosa. This would be feasible Inducing Ligand) following TLR-3 activation which alongside CD40 by understanding and exploring the antigenic inductive mechan- activates B cells in a T-dependent interaction to induce class- isms of the NALT in its unique form. Several lymphoid and switching to IgA+ B cells that produce sIgA43. It has been reported innate cells, e.g., T cells, B cells, macrophages, and DCs, have that CD8 + resident memory T cells (TRM) are generated by local been reported to be present in the NALT and are involved in antigen production at the respiratory mucosa following MHC-I the response not only to invading pathogens but also to vaccine restricted antigen presentation by stromal cells and migratory antigens40,41. Similar to the GALT, the DCs in the NALT are CD103 + DCs. These non-migratory cells remain in the lung tissue separated from the nasal cavity by a FAE lined with M cells and serve to respond during secondary infection, acting in both which facilitate the transfer of antigens to the underlying innate and adaptive fashion, owing to prestored cytokines such as Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 6 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. IFN-γ prestored at the transcriptomic level44. Indeed, targeting the When compared to the respiratory mucosa, the sublingual development of these cells by intranasal immunization could be mucosa has about 5 times relatively lesser surface area. In beneficial in controlling respiratory viruses. addition, the sublingual mucosa lacks antigen-sampling M cells, Follicular DCs and is devoid of organized lymphoid follicles. The oral cavity and the sublingual mucosa Nonetheless, several cellular populations have been reported in Although the oral cavity is near the nasal cavity, it is lined with the sublingual epithelium ranging from DCs of different squamous stratified epithelium which is a functional adaptation phenotype, to eosinophils, macrophages, mast cells, tissue- to withstand insults, prevent penetration of xenobiotic agents resident memory T cells, B lymphocyte and natural killer cells. and in turn contains diffuse lymphoid tissue as inductive In addition, CD11b+, CD11c+ EpCAM+ CD201+/− oral Langerhans machinery. Some areas of the buccal mucosa are keratinized cells possessing TLR 2,3,4,9 have been reported to be present—a such as the gingival, hard palate and the outer lips thus adding feature that increase antigen uptake and makes this route extra layer of imperviousness against insults and of course, desirable for vaccine administration47,48. antigenic uptake45,46. For this review, sites of possible antigenic induction, specifically the sub-lingual area will be discussed. This The vagina mucosa is due to the ease of accessibility and lack of keratinized The reproductive tract has received a relatively lower attention in epithelium which makes antigenic uptake easier than other sites mucosal immunity compared to the other mucosal routes. This of the oral cavity such as the gingivae, tonsillar areas and cheeks. region differs with respect to the innate and adaptive immunity, However, this region is continuously bathed with saliva, a feature mucus production and microbiota composition. Also, the vaginal that has limited drug and vaccine delivery through this route46. mucosal epithelium is stratified compared to the monolayer found npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 7 in the intestinal or lung mucosa49. The upper reproductive mucosa ClinicalTrials.gov/ NCT04674189 ClinicalTrials.gov/ NCT04668339 ClinicalTrials.gov/ NCT04566276 Other mRNA COVID-19 vaccine strategies with the delivery systems at different phases of clinical trials: FL full-length, IM intramuscular, LNP lipid nanoparticles, NA not available, RBD receptor binding domain (type-I mucosa) is enriched in sIgA while the lower reproductive mucosa (type-II) which encompasses the vagina and ectocervix References/Trial number lacks sIgA and are devoid of MALT. The region between the type-I &II mucosa is the transformative zone, which is rich in T cells and APCs, this zone also represents a frequent site of vaginal infection. The vaginal mucosa is covered by thick mucus which helps in trapping pathogens. The thickness of mucus and the composition of both immune and non-immune cells is influenced by hormonal 83,161 changes during the menstrual cycle. Generally, the submucosal 161 91 83 51 region of the type-I epithelium is rich in DCs, macrophages and memory lymphocytes while that of the type-II epithelium consists of a sparse network of DCs, macrophages and a few lymphocytes. Trial phase The uterine epithelium has a lymphoid aggregate of B cells and CD8+ T cells which becomes enlarged during infection50. Following the induction of vaginal infection, the effector site is I/II III II I I limited to the local reproductive mucosal immunity. This local vaginal immunity has been shown to be particularly important in providing full protection against HPV, HIV and HSV. Both Route neutralizing antibody (HPV) and T cells (HIV and HSV) are relevant IM IM IM IM IM in this context. This involves both CD4+ and CD8+ T cells as the former are required to drive a robust CD8 T cell response that mediates cytotoxicity of HIV-infected cells. Thus, the vaginal FL pre-fusion Spike Encoded protein mucosa should be explored for vaccine design against sexually transmitted diseases49. VEEV-FL-S-2P VEEV-FL-S RBD-S THE MRNA VACCINE PLATFORM NA Considerations in design and construction Synthetic mRNA vaccines are designed based on the blueprint of eukaryotic mRNA51. There are basically three types of mRNA Delivery system vaccines; linear conventional non-replicating mRNA, circular RNA Lipid-based and virally derived self-amplifying linear mRNA vaccines52,53. Of these, the two linear mRNA types are popularly employed in infectious disease vaccine applications. The two types have LNP LNP LNP LNP four essential features in common: a cap structure, 5′ and 3′ untranslated regions (UTRs), an open-reading frame (ORF), and a subunit, S spike protein, SA self-amplifying, VEEV Venezuelan equine encephalitis virus. 3′ poly (A) tail. An additional structure which is unique to the self- Conventional Conventional Conventional amplifying mRNA constructs is the genetic replication machinery mRNA type usually derived from Alphaviruses54. This replicase system is together formed by the four non-structural proteins (nsp), viz, nsp- 1 to nsp-4, all of which are encoded in the alphavirus genome55. SA SA The 5′ cap and 3′ poly (A) tails are crucial for enhancing the translational efficiency and transcript stability of the mRNA vaccine. In particular, the 5′ 7-methylguanosine (m7G) cap of Chulalongkorn University Imperial College London mRNA-based COVID-19 vaccines in clinical trials. Arcthurus Therapeutics the mRNA molecule plays a significant role in translation by blocking the 5′–3′ exonuclease-mediated degradation56. Similarly, poly (A) tail at its optimal length, may enhance translation and Curevac/Bayer provide protection to the mRNA.57. The 5′ and 3′ UTRs are critical AMS/Walvax Developers regulators of protein translation; hence, the choice and construc- tion of these UTRs are phenomenal for the optimal antigen expression and vaccine efficacy58. The coding sequence of the gene of interest may also be codon-optimised to synonymously substitute the rare codons with those codons whose tRNAs are abundant in the cytoplasm in order to achieve optimal produc- ChulaCov19 mRNA vaccine tion59. The mRNA construct can be modified by including codons such as pseudouridine, 2-thiouridine and 5-methylcytidine into the mRNA in a nucleoside-modified mRNA vaccine. This approach LNP-nCoVsaRNA has been shown to reduce innate recognition by PRRs and decrease the degradation of the mRNA molecules thereby increasing stability and translation efficiency59–61. Given the role ARCT-021 Vaccines CVnCoV Table 1. of the mRNA structural elements in determining vaccine efficacy, ARCoV careful considerations must be made in designing and construct- ing engineered mRNA vaccines. Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 8 ClinicalTrials.gov/ Purification for optimal expression Other mRNA vaccines against emerging/re-emerging viral diseases in preclinical and clinical development: CHIKV chikungunya virus, gB glycoprotein B, HCMV human cytomegalovirus, HIV human ClinicalTrials.gov/ Following the synthesis of the mRNA from the plasmid DNA, the resultant mRNA is often in complex with impurities which must be removed to liberate a purified therapeutic mRNA which is NCT03014089 NCT03325075 suitable as a vaccine candidate. The first purification step is to get References rid of other reaction components which are in complex with the 170,171 mRNA transcript by a series of extraction and precipitation 162 163 164 165 166 167 168 169 processes. The sample is left with shorter than normal mRNA arising from premature termination of the elongation step as well T cells and Neutralizing antibodies T cells and Neutralizing antibodies as longer than normal mRNA arising from linearized or non- T cells and neutralizing antibodies T cells and neutralizing antibodies T cells and neutralizing antibodies linearized template DNA14. Furthermore, the sample contains other contaminants such as residual DNA templates, enzymes and double-stranded RNA transcripts55. An effective and efficient Neutralizing antibodies Neutralizing antibodies Neutralizing antibodies purification of the mRNA to remove all these contaminants is crucial to generate a high-quality mRNA before it can be Protective response considerably utilized as a therapeutic substance. The elimination of the double-stranded RNA transcripts is of particular impor- tance as these can activate intracellular RNA sensors which triggers IFN-I production and reduces the downstream antigen- T cells driven adaptive response and efficacy of the mRNA vaccine62. In addition, purification of the mRNA is essential for optimal and efficient expression of the encoded protein which would enhance Subcutaneous and intradermal a sustained antigen availability, presentation, and downstream Intramuscular and intradermal Intramuscular and intradermal adaptive response. Following purification, the mRNA is further immunodeficiency virus, PC pentameric complex, LNP lipid nanoparticles, PC pentameric complex, prM-E pre-membrane & envelope. subjected to sterility, potency, identity, and purity testing before being finally formulated into a therapeutic product of Good Manufacturing Practice (GMP) grade. Thermal stability is a considerable issue with mRNA vaccine formulation as there is Intramuscular Intramuscular Intramuscular Intramuscular Intramuscular need to preserve an extensive cold chain from the development, transportation, distribution, and storage at all points along the chain. This remains a great disadvantage particularly for the Route developing countries where power supply is limited, and the cold chain maintenance is uncertain for distribution and storage – of the vaccines63. Autologous dendritic cells Immunogenicity and efficacy The immunogenicity of mRNA vaccines is driven mainly via the Delivery system expression of the encoded protein by utilizing the host cellular translational machinery thereby triggering robust cellular and Protamine humoral adaptive responses. The encoded protein antigen can be secreted, membrane-bound or retained in the cytosol depending LNP LNP LNP LNP LNP LNP on the mRNA sequence delivered into the intracellular milieu. This – sequence may be intrinsic to the primary antigenic protein or mRNA vaccines against some emerging/re-emerging viruses. engineered to either or not include both signal peptide and Hemagluttinin glycoprotein (H10N8 and H7N9) transmembrane domain sequences55. Although the innate immune cells are the desired target of mRNA uptake following in vivo delivery, however, there is possibility of uptake of the mRNA formulation by the cells at the site of injection which are non-APCs and since these cells possess MHC-I, they may HIV-1 Tat, Nef and Rev proteins translate and express the antigen in MHCI context. This off-target CHIKV structural polyprotein expression may result in cytotoxic T cell killing and ADCC, hence, Rabies virus glycoprotein efforts should be made to direct mRNA molecule to DCs to avoid Envelope glycoprotein off-target toxicity, improve uptake and enhance efficacy64. Similar to viral genomes, mRNA can be detected by intracellular nucleic Encoded antigen acid sensors such as TLR7 and TLR8 found in antigen-surveillant Fusion protein DCs, monocytes and macrophages59. These innate responses have gB, PC, pp65 been reported to contribute to the success of mRNA vaccines by driving both cytokine and chemokine responses which induce prM-E PrM-E adaptive cellular and humoral response against the encoded protein antigen55,65. Furthermore, the immunogenicity of mRNA vaccines can be linked to the provocation of both MHC-I and Emerging viruses MHC-II restricted antigen presentation. Chikungunya Being recognized by intracellular RNA sensors, exogenous mRNA has intrinsic immunostimulatory activity making it self- Influenza Dengue Table 2. adjuvanting, this property can however have both beneficial and Rabies HCMV Ebola Zika detrimental effect on the potency of the mRNA vaccine by RSV HIV triggering type-I IFN response with downstream immune response npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 9 on the one hand and impairing translation through the activation MRNA VACCINES AND EMERGING DISEASES of 2′−5′-oligoadenylate synthetase (OAS) and protein kinase R The mRNA vaccines against covid-19 (PKR) respectively60,66–68. Apart from optimal purification, there In the quest to end the current COVID-19 global pandemic, there are approaches which have so far been shown to further dampen have been concerted efforts worldwide to develop safe and the immunostimulatory activity and reduce the innate sensing of effective vaccines giving rise to many vaccine platforms being the mRNA thereby allowing a more efficient translation and developed and trialled in different parts of the world. This is increasing the immunogenicity of the mRNA construct, these are particularly important owing to the fact that there is no specific extensively covered in other reviews52,55,60,66,68–70. Self-amplifying therapeutic approach against the disease which led experts to mRNA (SAM) constructs are another strategy for increasing reach consensus that safe and effective vaccines are crucial in encoded antigen expression and improving immunogenicity at a order to put an end to the pandemic81. These efforts have led to comparatively lower dose than non-replicating mRNA since this an accelerated development of many next-generation vaccines can multiply in the intracellular milieu, a detailed discussion of the such as viral vectored vaccines, DNA vaccines, antigen- RNA species is covered in other reviews55,71–73. presenting cells and mRNA vaccines. So far, two mRNA vaccines have been approved for use clinically in many parts of the world, In vivo delivery systems for optimal translation and although there have been other next-generation vaccines expression currently in-use worldwide82. There are seven mRNA COVID-19 vaccine candidates in clinical An efficient delivery system is crucial for the success of mRNA trials as of January 2021 representing only 11% of the total 63 vaccines to reach their full potential in terms of translation, COVID-19 vaccine platforms in clinical trials. Out of these, only 2 immunogenicity and efficacy. It has been shown that expression mRNA-based vaccines being approved for human use so far83. The occurs and peaks within the first 4 hours following intravenous mRNA vaccines currently in use were developed by Moderna administration of mRNA vaccine and this may persist up to (mRNA-1273) and Pfizer-BioNtech (BNT162b) US companies. Both 10 days after intramuscular or intradermal administration74. Many vaccines consist of a nucleoside-modified mRNA encoding the strategies have been explored for in vivo delivery of mRNA into SARS-CoV2 spike protein with two proline mutations in the target cells. These strategies aim at ensuring the protection of the sequence at positions 986 and 987 to keep the encoded protein mRNA molecules from degradative actions of nucleases, maximal locked in the pre-fusion conformation. This modification is delivery of the mRNA into cells, effective translocation into the necessary to mimic the native three-dimensional conformation cytosol from the endosomal compartments and improving the of the SARS-CoV2 spike protein just before binding to the ACE2 overall translation, protein expression as well as immunogenicity receptor of host cells. The mRNA is nucleoside-modified by uridine of the mRNA. These strategies include the use of lipid-based, replacement with 1-methylpseusouridine to reduce innate polymer-based or hybrid carriers as delivery vehicles. The most responses as discussed earlier. The delivery vehicle used in both commonly explored delivery vehicle with a track record of high cases is the LNP-based vehicle and both vaccines are administered efficiency is the use of lipid-based carriers which may be in form of parenterally in 2 separate doses 28 days apart and 21 days apart lipoplexes or lipid nanoparticles (LNP)52. Lipoplexes are complexes for Moderna and Pfizer vaccines respectively. The immunogenicity formed by the electrostatic interactions between cationic lipo- and safety profiles have been good for both vaccines; however, some carriers and RNA molecules. Liposomes are nanosized the Pfizer vaccine reportedly has a slightly higher efficacy than the spherical vesicular structures made of amphiphilic phospholipid Moderna vaccine with a record efficacy profiles of 94.5% and 95% bilayers composed of a hydrophilic head and two hydrophobic for Moderna and Pfizer vaccines respectively81,82,84. Overall, the tails with an aqueous core. The spherical structures assemble two vaccines have shown protective cellular and humoral when the phospholipid bilayer is present in an aqueous responses targeted towards the SARS-CoV2 spike protein with environment, whereby the hydrophilic head tend to interact with the T-cell response biased towards the Th1 response. Antibody the aqueous environment while the hydrophobic tail tend to induction is reportedly broadly neutralizing with titres higher than interact with one another75. They have been used as vehicles for that of human convalescent serum from COVID patients. There is gene delivery, silencing-RNA delivery and have been explored in no significant difference in the humoral response of the elderly both cancers and infectious diseases for mRNA vaccine delivery. when compared to that of the younger population; this is of This is due to the high transfection rates of liposomes compared particular importance since the older populations develop more to other non-viral vectors, their biodegradability and ease of severe diseases82,85. However, recent vaccine effectiveness studies synthesis. In the field of IVT mRNA delivery, cationic lipids like revealed a reduced effectiveness in the elderly (≥65 years) compared to younger adults (18–64 years) with an adjusted VE 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) of 79.8% and 95.1% respectively86. The main disadvantage of and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) have these two vaccines is that they have to be stored at a very low been explored as well as 1,2- dioleoyl-sn-glycero-3-phosphoetha- temperature to ensure the stability of RNA along the supply chain. nolamine (DOPE) which is a zwitterionic lipid69,76,77. Ionisable Despite its higher efficacy and lower cost, the Pfizer vaccine lipids which are neutral at physiological pH but become cationic in initially required a much lower storage temperature of around an acidic pH remain viable alternatives to cationic lipids offering a −80 to −60 °C and poses more storage challenges as compared to relatively reduced toxicity and facilitate endosomal escape of the Moderna vaccine which can be stored at a relatively higher antigens for cytosolic delivery78. LNP-based delivery systems are temperature of about −25 to −15 °C which is more feasible. More the most used carrier vehicles at present. Many studies have recently, a warmer temperature storage between −25 and −15 °C reported their successes in both infectious diseases, e.g., zika virus, has also become feasible for Pfizer vaccine in a duration of up to influenza virus, HIV and rabies virus infections as well as in cancers. 2 weeks following which the vaccines can be returned to the Notably, LNPs have so far offered the best results. The basic ultracold temperature for a more prolonged shelf life87. Both components are cationic/ionisable lipids, cholesterol, polyethylene vaccines have been reported to cause mild adverse reactions such glycol (PEG) lipids and phospholipids; these work synchronously as soreness, redness or pain at injection sites, fever, headache and to achieve efficient protection, uptake, endosomal escape, and rare anaphylaxis. Overall, the incidence of adverse events is lower translation of mRNA molecules to drive robust cellular and in the Pfizer than the Moderna vaccine84. Vaccine effectiveness humoral responses52,55,78–80. Polymer-based and hybrid delivery (VE) studies to assess the real-world impact of these vaccines have systems are extensively discussed in other reviews52,68,77. also revealed positive impact in preventing hospitalization and Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 10 severe diseases. A study involving 21 US hospitals revealed an routes of mucosal vaccination which induce varying degree of effectiveness in preventing hospitalization standing at 88% and vaccine-specific immune responses that in-turn determines the 93% for Pfizer and Morderna vaccines respectively after a 2-dose vaccine efficacy. Mucosal vaccines have been explored through schedule. This was relatively higher than that obtained for the the oral, nasal, sublingual, ocular, rectal and vaginal routes of Jansen vaccine which was 71% effective in preventing hospitaliza- administration. However, the oral and nasal routes have been tion. Apart from the fact that the Morderna vaccine has a higher mostly employed since these induce a broader mucosal and VE than the Pfizer vaccine, this study also noted a decline in the VE systemic responses compared to the other mucosal routes93,96. of Pfizer vaccine within 120 days. A higher anti-RBD antibody was The nature of the antigen, the targeted mucosal tissue and the also reported for Morderna than the Pfizer vaccine. These desired site of the induced immune response largely influence differences in VE were possibly linked to the amount of mRNA the selected routes of vaccination97. Since different mucosal particles in the Morderna vaccine and a longer interval of 28 days routes induce different level of responses in terms of potency between doses as compared to the 21 days of Pfizer88. Another and longevity, this reflects the variation in the cellular make up study reported a lower overall VE of 86% for immunocompro- and the organization of the mucosal lymphoid tissues at the mised individuals in reducing hospitalization and severe diseases different mucosal sites. Hence, it is expected that the same as compared to immunocompetent individuals after 2-dose vaccine/antigen administered through different mucosal routes vaccination with either Morderna or Pfizer89. However, these would result in varying degree of protective immunity93,98. For vaccines are less effective in preventing infection especially example, it has been shown that vaccination against Mycoplasma in the face of evolving viral strains. gallisepticum induces a better effectiveness via the ocular route In addition to the approved mRNA COVID-19 vaccines described than the nasal route, whereas, the oral route of administration above, the other mRNA-based COVID-19 vaccines under clinical has a relatively negligible vaccination outcome99. This is so studies are summarized in Table 1. because effector T cells and B cells preferentially home to the inductive mucosa compartment and the neighbouring anatomi- mRNA vaccines in other viral diseases cal sites. The route of vaccination needs to be carefully considered depending on the mucosal tissues targeted by the mRNA vaccines are potentially beneficial as compared to other pathogen in question. conventional vaccines in the fight against emerging infectious diseases as they can be rapidly developed and optimized in a scalable manner. This enables them to be rapidly directed against The nasal route the most rapidly emerging and re-emerging pathogens, most The intranasal route has been shown to induce robust cellular and especially viruses, which are potential causes of epidemic and humoral response at the respiratory tract against different pandemics52. In contrast, conventional vaccine development is pathogens following vaccine-specific induction of the nasal rather time-consuming which could take several years to develop immune system/NALT100. In addition, nasal vaccination effectively and often complicated by scale-up and commercialization issues induces response in the bronchial-associated lymphoid tissue while mRNA vaccines can be effectively developed within a (BALT), the urogenital tract, the salivary gland and the gastro- shorter time frame to meet large-scale demand. Furthermore, intestinal tract93,101. Intranasal administration of influenza vaccine mRNA vaccines can be rapidly modified and developed to be (Flumist) has been shown to display a superior protective efficiently directed against emerging and fast-mutating pathogens immunity than the intramuscularly administered counterpart with in response to outbreaks since they are not prone to issues of pre- effective IgA production at the nasal epithelia94,102. In addition, existing immunity which remains a huge challenge to the success this has been reported to provide cross-protection against of viral vector-based vaccine90. There are several pre-clinical seasonal influenza strains including genetically drifted strains103. studies of mRNA vaccines focusing on emerging viral diseases Furthermore, the intranasal route has been explored to induce such as Zika virus, HIV, CMV, Influenza virus, Ebola virus and Rabies protective immunity at the GIT against shigellosis. Preclinical and virus with encouraging results in terms of safety and effective clinical trials revealed a potent GI protection following intranasal immunity conferred by both CD4 and CD8 T cells, as well as vaccination against Shigella flexneri thereby further indicating the neutralizing antibodies. Some have subsequently been translated crosstalk between the different mucosal sites104. In addition, the to clinical trials in human subjects68. Although both non- crosstalk between the nasal and genital mucosa is being replicating and self-amplifying vaccines against viral diseases researched to tackle genital infections and sexually transmitted have been subjected to pre-clinical and clinical studies, the diseases via intranasal vaccination. Since the nasal route of conventional mRNA vaccines have so far progressed furthest in vaccination induces robust antibody response in the vaginal clinical practice91. Summarized in Table 2 are some of the most mucosa, this strategy has been explored with success in HIV important virus-directed mRNA vaccine trials in either preclinical research. Here, preclinical studies in rhesus macaques showed the or clinical studies. induction of protective IgA and IgG at the vaginal mucosa against HIV-1 infection and subsequent prevention of HIV transmission via the genital tract after an intranasal immunization with a trimeric EXPLORING THE DIFFERENT MALT COMPARTMENTS— HIV-1 gp41 protein grafted on a virosome105. However, this MUCOSAL ROUTES OF VACCINATION strategy induced a relatively low T-cell response at the vaginal The concept of mucosal vaccination was borne out of the mucosa as compared to the direct vaginal vaccination, hence, a realization that greater than 90% of the pathogen that infect synergistically broader response encompassing both humoral and human enters the human system via the mucosal routes and the cellular anti-HIV response was achieved by combining intranasal limitation of the current injection-based immunization in immunization with a local vaginal vaccination in a prime-boost substantially controlling mucosal infection and systemic dis- technique106. This technique is further necessitated by the failure semination of these pathogens5,92,93. In general, the mucosa has of the vaginal immunization with HIV gp140 to induce protective a highly organized immune system thereby rendering it an immunity in healthy women in a clinical trial107. Therefore, apart important target for vaccination93,94. This is reinforced by the fact from the potential use to control respiratory pathogens, mRNA that priming of DCs at a specific mucosa largely determine the vaccines can be targeted in a prime-boost immunization strategy homing of T cells to the same mucosa and other distant mucosal through intranasal and intravaginal administration to tackle HIV, sites to fight pathogens at these entry portals95,96, based on the HPV and other STDs. In a recent study, a parenteral prime principle of common mucosal immunity. There are different immunization with mRNA SARS-CoV-2 vaccine followed by an npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 11 Above 90% in most part of intranasal boosting with an adenovirus-vectored (mRNA prime-Ad . CTB cholera toxin subunit B, CTL cytotoxic T-lymphocytes, HA Mucosal IgA and systemic IgG neutralizing 70–90% (severe disease) Mucosal IgA and systemic IgG neutralizing 70–90% (severe disease) Above 50% but variable Variable in adult, above boost) vaccine significantly induced a very good IgG, IgA and TRM Variable but generally Strong gut protection Strong gut protection at the respiratory mucosa. When compared with a parenteral DNA prime-intranasal Adenovirus boost, the mRNA prime-Ad boost 85% in children Vaccine efficacy strategy had a more efficient neutralization of the virus variant of above 70%172 above 85% above 85% concern and a more comprehensive T cell response comprising the globe of both circulating T cells and TRM44. Indeed, the nasal route of vaccination offers several advantages. Apart from the induction of broad humoral and cellular responses, the nasal immunization – strategy avoids first-pass metabolism due to abundant venous Gut antitoxin and mucosal IgA, anti-LPS systemic neutralizing IgG. CTL probable blood that delivers antigens directly into the systemic circulation, HA and NA-specific mucosal IgA and mucosal IgA and anti-LPS antibodies limits the risk of anaphylaxis, offers a very large surface area, and CTB-specific antibody, gut antitoxin, Enteric coated capsule Mucosal IgA, systemic IgG and CTL avoids the harsh PH and the excess degradative enzymes Enteric coated tablet Serum neutralizing antibodies173 Systemic IgG and mucosal IgA attainable at the oral mucosa. In addition, this route offers a dose-sparing advantage. However, this route poses the risk of Vibriocidal antibodies172 rapid antigen clearance due to the mucociliary actions leading to short nasal residence time. There is also a poor uptake of soluble Protective immunity antigens and antigen uptake may be impaired in patients with respiratory problems94,101. antibodies antibody antibody The oral route 2,33,92,93,95,122 The oral route of vaccination is capable of inducing robust immune response at the GIT, salivary gland, mammary gland as well as the NALT93. Along the GI system, effector immune cells are preferentially home to the lamina propria, salivary gland, A summary of the mucosal vaccines platform with the specific routes and the induced immune responses. Adapted from refs. stomach and intestinal epithelium with a robust sIgA response. Route of administration Dosage form In addition to the broad mucosal immunity, the oral route has been shown to elicit a systemic neutralizing IgG response92,94. The oral route remains the most patient-friendly route of Liquid Liquid Liquid Liquid Liquid Liquid Spray therapeutic intervention with over 60% of pharmaceutical drugs currently being administered orally. However, the success of an oral vaccination depends on the effective delivery of the antigen to the intestine, crossing of the intestinal epithelium and subsequent activation of the intestinal APC5. This makes the oral route the most challenging in terms of vaccine development partly due to the harsh mucosal environment which rapidly degrades the antigen and partly due to the immunological Nasal tolerance which fails to induce protective immunity, in addition Oral Oral Oral Oral Oral Oral Oral Oral to the mucus barrier and flexible residence time determined by hemagglutinin, IgA immunoglobulin A, IgG immunoglobulin G, LPS lipopolysaccharide. gastric emptying. This has led to several experimental attempts of oral vaccination which failed to make it past clinical trials due to disappointing results5,92. Nevertheless, there exist some oral vaccines with good clinical efficacy; these include the Biopolio oral polio Shanchol, Euvicol vaccines against polio, rotavirus and cholera. These vaccines are vaccine (bPOV) licensed by the WHO and remain a good example for the Trade name outstanding efficacy of oral vaccination giving a strong prospect Vaxchora Duchoral for oral mRNA vaccination94. The oral route also offers many Rotateq Flumist Rotarix Vivotif advantages over other routes. It is a relatively safe and cost- effective route allowing for self-administration thereby increas- – ing vaccine acceptance and facilitating vaccine distribution. Live attenuated polioviruses Live attenuated type 4 & 7 Furthermore, there is no risk of needle-stick injury or local Inactivated Vibrio cholerae Inactivated Vibrio cholerae Currently licensed mucosal vaccines. injection-site reaction92,94,95. Nature of vaccine The rectal route Live reassortant Live attenuated Live attenuated Live attenuated Live attenuated serotype 1 & 3 Although the oral and nasal routes are the most explored, researchers are now exploring other mucosal routes such as the with CTB Licensed for use in the military. sublingual, rectal and the intravaginal routes. The rectal immunization route induces protective response at the rectal mucosa, the nasal secretion and the tears. This route is suited for Salmonella typhimurium pathogens which enter through the GIT and particularly those adapted to the lower GIT93,94. The administration of a peptide- vaccinia prime-boost vaccine via the intrarectal route in Influenza virus Vibrio cholerae Vibrio cholerae Vibrio cholerae macaques was shown to elicit a high avidity CD8 T-cell response Adenovirusa at GI mucosa and this correlated with a lower dissemination of Poliovirus Pathogen Rotavirus Rotavirus Table 3. SIV108. In addition, earlier studies have shown that intrarectal immunization was more effective than the subcutaneous route in a the control of SIV viraemia or mucosal infection and the Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 12 induction of CD8 T cells in macaques109. Perhaps, this route may CONSIDERATIONS FOR MUCOSAL DELIVERY OF MRNA be explored for mRNA vaccine delivery against HIV. VACCINES The prospects The sublingual route Virtually all the mRNA vaccines currently being developed against The sublingual has gained a lot of research attention in recent infectious diseases are designed for parenteral administration years. This route has been shown to also induce a very broadly mainly via the subcutaneous or intramuscular routes. Notably, the disseminated mucosal and systemic responses. Studies have also licensed COVID-19 mRNA vaccines described above are also given depicted a robust systemic IgG and mucosal IgA induction as well parenterally. Although parenteral administrations of vaccines have as CD8 T-cell response following sublingual vaccination110,111. been generally protective as they generate effective systemic Furthermore, a comparison of the sublingual vaccination with immunity, they often induce a relatively weak local mucosal intranasal, transdermal, intravaginal and intramuscular vaccination immunity which may be inadequate for the complete elimination using the HPV 16 L1 protein depicted a more robust immunity of the pathogens from mucosal sites6. Furthermore, a parenterally from the sublingual routes than the other routes111. This route has immunized individual may develop a persistent infection on the comparable effectiveness to the nasal route in terms of the mucosal surfaces, owing to inefficacious local mucosal immunity; breadth of the induced response and in contrast to the nasal route, this may subsequently be transmitted to unimmunized or there is no risk of perturbation of the CNS by the redirection of immunocompromised individuals118. However, an effective muco- antigens or adjuvants112. Since the antigen is directly administered sal vaccine is capable of inducing both potent mucosal and via the oral mucosa into the bloodstream, the sublingual route systemic immunity through a robust humoral immunity, mediated bypasses the action of proteases and the harsh GI environment, by sIgA (as discussed earlier), and cell-mediated immunity33. In thereby, making it a very promising route of mucosal administra- addition, IgG is also produced at the mucosal surface following tion94. The sublingual route has equally been shown to induce mucosal vaccination to play role in the neutralization of protective immunity in the lungs and the GIT. A study depicted a pathogens but the IgG concentration is relatively lower as they protective response against influenza virus challenge in mice are rapidly degraded in the mucosal environment6. Mucosal following sublingual vaccination with a live attenuated or formalin- vaccination also induces both long-term memory B and T cells via inactivated influenza virus113. Sublingual vaccination with a homing of effector and memory cells to the mucosal sites from recombinant Helicobacter lipoprotein provides a protective the draining lymph node through the blood to the mucosal sites. immunity against Helicobacter pylori in the stomach, more so, this As shown earlier, this is achieved through the homing receptors route was better than intragastric vaccination in terms of the CCR9 and CCR10 expressed by the adaptive immune cells and are protective response114. Hence, provided a perfect delivery vehicle acquired in the lymph node33. The mucosal response to vaccines, can be constructed, this route has a potentially wide applicability like pathogenic stimulation, is largely driven by the presence of for mRNA vaccination against a wide variety of pathogens. MALT at mucosal sites with inductive sites rich in abundance of APCs, B cells and T cells as described earlier. In addition, the The vaginal route specialized M cells at the epithelial surface actively trap mucosal vaccine antigens for delivery to the underlying immune cells of The vaginal route is a potentially viable route for sexually the MALT at the submucosal region100. No doubt, mRNA vaccines transmitted diseases such as HIV. The vaginal mucosa has a large would benefit from these immunological advantages if adminis- surface area which has a relatively low level of degradative tered via the mucosal route thereby fostering an intense enzymatic activity with a possibility for self-administration of protection at the mucosal portal of entry of the emerging viruses vaccines94. However, this route may induce a relatively weak to limit infection and transmission. mucosal response which is most probably limited to the local vaginal mucosa. Furthermore, immunization with live or vector vaccines induce effective mucosal response while vaccination The challenges with inactivated or subunit vaccines have shown poor responses The comparative advantages offered by mucosal vaccination in in mice96. For example, it was depicted in a study that terms of broad range of immune protection spanning the mucosal intravaginal immunization with Salmonella enterica expressing and systemic immunity, including the non-immunological advan- the HPV16 L1 protein induced a protective mucosal and systemic tages offered in terms of patient’s compliance as well as the humoral and cellular responses which were protective against absence of local reactions form a very strong basis for the HPV and prevent the development of subcutaneously trans- consideration of mucosal delivery of mRNA vaccines. However, planted HPV16 tumour115. Furthermore, the intravaginal route mRNA vaccines, like other vaccines, are confronted with many can be explored as an adjunct to other immunization routes to challenges at the mucosal surfaces which must be overcome improve vaccine response. For example, intravaginal administra- before exploiting the full potential of mucosal vaccination. For tion of TLR3 and/or TLR9 agonists following subcutaneous example, the tolerability issues remain a cogent reason why all the immunization with HPV E7 antigen lead to about fivefold increase currently licensed mucosal vaccines are either live attenuated in the level of INF-gamma-producing CD8 T cells homing to the (Vibrio cholerae, Influenza, Poliovirus, Salmonella typhimurium and vaginal mucosa with a threefold higher level of tumour regression Rotavirus vaccines), whole-cell inactivated (Vibrio cholerae vac- of HPV16 tumour compared to only subcutaneous immunization cines) or live re-assortant viruses (Influenza, Rotavirus and in mice116. However, it is worth noting that intravaginal Salmonella typhimurium vaccines). These are efficient in breaking immunization may be impaired by the menstrual cycle driven the tolerance through the induction of robust inflammatory by the estradiol level. It has been shown that elevated estradiol response95. Also, at the respiratory mucosa, the mRNA vaccine, like level induces vaginal mucous secretion which in turn reduces other vaccines, faces challenges discussed earlier which are further antigen penetration of the vaginal epithelium, prevented CD8 compounded by mucosal RNAse degrative actions and epithelial T-cell priming and inhibited antigen uptake by APC across the barrier that impedes mRNA uptake18. In addition, the mRNA vaginal epithelium following intravaginal immunization. This vaccine faces challenges relating to salivary dilution, epithelial effect is reversed following the removal of the estradiol-induced barrier and immunotolerance at the sublingual mucosa while mucous barrier with mucinases117. Indeed, this route has a those of the vagina and rectal mucosa are due mainly to mucus potential in mRNA immunization for the control of HIV and other and epithelial barriers as shown in the next figure. These hurdles urogenital pathogens, either alone or in combination with other are the main limiting factors against the exploitation of mRNA immunization routes. vaccines via the mucosal route; therefore, there is need to develop npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 13 strategies to overcome these mucosal barriers thereby ensuring which can multiply and allows persistent antigen presence. In this effective delivery and efficient uptake of the mRNA molecules by regard, a self-amplifying mRNA could be considered since the mucosal APCs to induce immune response in the MALTs. mRNA construct can multiply several times, owing to its inherent design as discussed earlier, although this requires the mRNA molecules to be delivered directly into the mucosal epithelial cells. The existing currently licensed mucosal vaccines: lessons for This means that a delivery system is required to protect the sa-RNA mucosal mRNA vaccines molecules before uptake and intracellular delivery. For the Although there have been several attempts to develop mucosal conventional mRNA, a delivery system that closely mimics a live vaccines over the years, only a few of such vaccines have been pathogen in terms of innate stimulation should be designed by licensed commercially for human use. Many vaccines that showed including PRR ligands since it is practically impossible to have a good results in animal models often failed in clinical trials. Owing multiplying delivery vehicle. Nonetheless, inactivated vaccines are to the difficulty of the mucosal microenvironment, most of the also devoid of multiplicability and some mucosal vaccines with licensed vaccines are live attenuated to enable multiplication and good efficacy are inactivated. Therefore, constructing the delivery induction of broad immunity. There are also licensed inactivated system to include a microbial ligand as the case with the inclusion vaccines against Vibrio cholerae which are less immunogenic but of CTB in Duchoral could help mimic inactivated vaccines and relatively circumvent the safety issues associated with live induce durable response. Overall, as shown in the table above, the vaccines5,33. Furthermore, most of the licensed vaccines are vaccine-induced response with all the mucosal vaccines is driven approved for oral use while only one is approved for use through by sIgA mucosal antibodies as well as systemic neutralizing IgG. For the nasal route33. The live vaccines consist of attenuated bacteria example, in the case of the licensed intranasal influenza vaccine, or viruses that are made less virulent than the wild-type pathogen. nasal IgA has been reported to be crucial for vaccine efficacy in These live attenuated vaccines have the dual advantage of children and this correlates with protection in human experimental intrinsic adjuvanticity and high antigenic availability. In addition, challenge studies. In addition, T-cell response and innate immunity these vaccines can be in the form of vectorized live vaccines that are important contributors to protective immunity in a similar way consist of live viruses or bacteria that express the recombinant to immune response induced by the wild-type virus, a strong antigen from the pathogen of interest33,95, however, these indication that site-specific immunization is key to have a response vectored vaccines may be relatively less effective because of comparable to natural infection44,124. Furthermore, oral polio possible pre-existing vector immunity as seen with adenoviral vaccine (OPV) containing a mixture of different live attenuated vectors. The live vaccines may set up a local infection or be poliovirus serotypes has been reported to induce protection via engineered to replicate in the mucosal environment to increase both systemic and humoral immunity when administered at a the antigenic load, an attribute that is relevant to overcome complete dose. The systemic IgG response is effective in the mucosal tolerability119,120. One main challenge associated with preventing neuroinvasion and avoid paralytic symptoms. The the live vaccine is the balance between attenuation and gastrointestinal IgA, which is unique to OPV compared to the immunogenicity. Attenuation could be achieved either by serial injectable inactivated polio vaccine, is relevant in preventing passages or molecular modification such as gene deletion. A person-person transmission since these antibodies control multi- trade-off exists between these two important properties such that plication at the infection site (GIT)5. This is a strong indication that an effectively attenuated vaccine is safe but less immunogenic mucosal mRNA vaccine administration could indeed a robust and vice-versa33. A relatively safe and highly immunogenic response which would be strongly preventive against infectious vaccine is obtainable via molecular attenuation by precise gene colonization and/or systemic dissemination. Strategies to enhance mutation as seen with the case of Salmonella vaccine121. Another the successful utilization of mucosal mRNA vaccines are sugges- drawback of the live vaccines is the possibility for reversion to the tively discussed later in this review. virulent strain and the reactogenicity which pose a huge safety risk, especially in the immunocompromised, elderly or infants93. These safety issues are the main driving force for the considera- Mucosal mRNA vaccine delivery vehicles: prospects from pre- tion of non-living inactivated whole-cell or subunit mucosal existing experimental studies vaccines. There are currently 3 licensed inactivated vaccines In the context of mucosal exploitation, mRNA vaccines have been ((Dukoral, Shanchol and Euvichol) produced by inactivating the experimentally utilized mainly via the intranasal routes as well as native pathogen with heat, chemical or radiation. Since these intravaginal administration125, there is however no known oral cannot multiply, they are generally safer than the live vaccines, mRNA vaccine administration till date,118 most probably due to however, they are less immunogenic and therefore require the extensive challenges posed by the GI mucosal degradative booster doses5,33,93. The subunit vaccines are generally much environment. The nasal route is widely considered for mucosal safer than the live or inactivated vaccines, but they are much less delivery because it presents fewer challenges than the oral route immunogenic, and their success have been limited due to the due to the non-abundant secreted enzymes, lack of acidic rapid degradation, poor uptake and the barriers in the mucosal environment, smaller surface area and lesser dose requirement environment. There is no licensed subunit mucosal vaccine, but compared to the oral route42. In addition, the nasal mucosa the cholera toxin B (CTB) subunit is the only subunit antigen similarly possesses the nasal associated lymphoid tissue (NALT) to be included in a licensed vaccine as an adjunct in the whole cell which is rich in APC, B cells and T cells overlaid by M-cells which killed cholera vaccine (Dukoral) due to its binding affinity to facilitate antigen capture and delivery18,42. There is a number of receptors on the mucosal epithelial cells and its high immuno- studies in which different delivery vehicles were explored with genicity95,122,123. The currently licensed mucosal vaccines in success in mucosal mRNA vaccine delivery. An early study in clinical use are summarized in Table 3 alongside their immuno- 2010 showed that intranasal administration of naked mRNA- genicities and efficacies. Hsp65 which encoded the Hsp protein of Mycobacterium leprae induced protection against virulent Mycobacterium tuberculosis Lessons. In the context of mRNA vaccine mucosal administration, infection in an experimental mice 30 days after vaccination. There some crucial lessons could be gathered from the perspective of was robust production of Th1 cytokines namely interferon- licensed vaccines. First, it is imperative to note that most of the gamma and TNF-alpha. The study further depicted the role of vaccines are live attenuated owing to the mucosal tolerance. lung APC in capturing the mRNA molecules starting within 30 min Hence, it is worth considering when designing an mRNA vaccine and lasting up to 8 h post-vaccination126. However, in a more formulation to have a construct that closely mimics a live pathogen recent study, the role of an intranasal delivery system in driving a Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 14 more robust response compared to naked mRNA was depicted. In specific cellular responses and effective tumour size reduction in a this study, an increased nasal residence time of the mRNA therapeutic setting132. Overall, there are prospects from the molecules was achieved using a complex of cyclodextrin, a experimental trials of mucosal delivery vehicles in favour of bioadhesive sugar molecule, coupled with low molecular weight the mRNA vaccine. Of course, there exists a huge research gap in polyethylenimine (PEI-2K) for the delivery vehicle of mRNA-gp120 the field of mRNA mucosal vaccine delivery, hence, more encoding the gp120 antigen of HIV. Results from this study extensive research is required to further unravel the potential of revealed that the CP2K-mRNA increased transfection efficiency in mRNA vaccines and to accelerate this success to clinical cell lines in-vitro. More importantly, intranasal administration in applications in mucosal vaccination. mice prolonged the residence time of mRNA in the nasal mucosa owing to the mucoadhesive property of cyclodextrin, and Innovative strategies to improve mucosal immunity enhanced crossing of epithelial barrier by paracellular and transcellular transport with higher safety profile compared to Efforts towards the improvement of mucosal vaccines have been high molecular weight PEI25k-mRNA system. In addition, CP2k- focused on the development of novel delivery strategies which mRNA showed a more robust HIV gp120-specific cellular and can overcome one or more of the challenges encountered at the humoral responses than naked mRNA or PEI25k-mRNA, this is mucosal environment with the sole aim of preserving antigen depicted by comparatively higher systemic IgG and mucosal IgA structural integrity, enhancing antigen bioavailability, and achiev- at both nasal and vaginal mucosa, higher CD8+ and CD4+ ing successful induction of both mucosal and systemic immune cellular responses with a balanced TH1/TH2/TH17 cytokine responses. These strategies vary depending on the targeted responses18. In further studies, CP2k demonstrated an enhanced inductive site and mucosal compartment92,93. The nature of the trafficking of mRNA molecules to the lymph node and triggered a antigen formulation is a crucial determinant of the enhancing more potent maturation of DCs in vivo after intranasal adminis- strategy to employ. Soluble vaccine antigens encounter impeded tration of CP2k-mRNA encoding OVA antigen when compared to uptake by the mucosal immune cells due to the constantly CP600-mRNA (containing 600dalton PEI) and PEI25k-mRNA renewing mucous barrier which limit antigen uptake. This issue (without cyclodextrin)127. Sugar modification of LNP has been can be overcome via the use of mucoadhesive molecules such as shown to enhance mucosal mRNA delivery. In a study on chitosan and starch which functions to allow a close contact Influenza A H1N1 virus using cationic LNP delivery system, the between the antigen and the mucous membrane, thereby, protein expression in-vitro was relatively higher when the LNP increasing soluble antigen uptake after enhanced adhesion of was modified with mannose (LNP-man), a ligand of innate the antigen to the mucosal surface. Chitosan has been shown to receptors, compared to the unmodified counterpart. Further- have a dual function of acting both as a mucoadhesive agent and more, protective humoral and cellular responses were reported as an adjuvant, hence it increases uptake and immunogenicity, following intranasal administration of 2 separate doses each of perhaps sufficient to break the mucosal tolerance. In addition, it is mRNA-H1HA-LNP and mRNA-H1HA-LNP-man encoding Influenza possible to formulate an additional adjuvant together with H1N1 HA protein. Although, both were effective in preventing chitosan with the aim of inducing robust immunity. Chitosan- weight loss and death in mice challenged with lethal dose of based delivery systems have been explored with success for H1N1 virus two weeks post-immunization, the response was intranasal and intravaginal vaccination33,133,134. On the other slightly higher in the LNP-man group signifying that ligand hand, particulate vaccine antigens are generally more successful modification of delivery systems can drive a more potent than the soluble antigens. This approach involves the encapsula- response via the mucosal route128. tion or intrinsic formulation of the antigens in form of particles The intranasal route has also been explored in the delivery of which prevent rapid degradation of the antigens and can, at the mRNA encoding neutralizing antibodies against some infectious same time, be manipulated for targeted delivery of antigen to APC diseases. In one study, mRNA-encoded neutralizing antibodies and M-cells or to include adjuvants in the vaccine formulation. achieved in vivo neutralization of RSV with minimal inflammation This includes the use of polymers, Virus-like particles, biodegrad- following aerosolized administration into the lungs. Furthermore, able microparticles such as Poly (lactic-co-glycolic acid) (PLGA) or GPI-anchored mRNA-encoded neutralizing antibodies were nanoparticles such as liposome and bacterial ghosts. This strategy retained on the plasma membrane of lung epithelial cells after allows for a controlled and/or continued release of antigen while expression and the neutralizing activity of the antibodies were also preventing the degradation of the antigen and adjuvant effective against RSV with or without GPI anchor, a promising before reaching the target site. It is also possible to include basis for mRNA-based pulmonary prophylaxis129. A similar result adhesive molecules such as lectin which offers the function of was reported in another research following intravaginal adminis- increasing residence time by bringing into contact the antigen tration of aerosolized mRNA encoding neutralizing antibodies and adjuvant with the mucosal surface. PLGA microspheres against HIV gp120, this similarly achieved expression of the anti- strategy allows 2 types of vaccine release, viz, controlled release HIV gp120 neutralizing antibodies in the genital epithelium in whereby vaccine antigens are continuously released mimicking sheep model130. There are few studies reporting intranasal continuous boosters, and pulsatile release whereby vaccine administration of mRNA-based cancer vaccines leading to the antigens are released in two distinct time intervals mimicking regression of tumour growth. A research group reported that the 2-dose booster strategy33,135. The VLP is currently being explored intranasal administration of an mRNA-based cancer vaccine as a mucosal delivery strategy. Novel techniques include the use encoding chicken OVA (mRNA-OVA) delivered with a nanoparticle of the VLP to co-deliver antigens together with an adjuvant. In a carrier showed potent prophylactic and therapeutic efficacy recent study, a chimeric VLP was bioengineered to express HA against tumour growth and progression in mice which were more influneza antigen and coated with the protozoan Giardia lamblia potent than observed with naked mRNA-OVA or the negative surface protein, variant-specific surface protein (VSP). The VSP control (mRNA-GFP). The result from this study also revealed successfully protected the VLP system from degradative enzymes robust OVA-specific CD8 T cells as the main driver of the tumour and PH fluctuations and provided an additional adjuvanting immunity and was present in the mRNA-OVA nanoparticle properties. Oral immunization of mice with this chimeric VSP- system131. A similar result was observed recently in another coated VLP expressing the influenza HA antigen induces a research following intranasal vaccination of mice with mRNA-CK19 remarkable immune response and protected the mice following vaccine encoding cytokeratin-19 which was encapsulated in a challenge with Influenza virus136. Lipid-based polymers represent cationic liposome-protamine complex (LPC) delivery vehicle. A another important strategy for particulate delivery of mucosal robust antitumor immunity was reported with abundant tumour- vaccines. The lipid-based polymers that have been explored npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 15 include liposomes, bilosomes, virosomes and proteosomes and engineered to express DC ligand and were shown to induce immune-stimulating complexes (ISCOMs). The proteosomes con- mucosal immunity that was protective against lethal Bacillus sist of Neisseria species-derived hydrophobic proteinaceous anthracis challenge following oral immunization94,96,142,143. As nanoparticles which are capable of associating with the hydro- seen with other routes of administration, the immune response phobic domains of antigens and enhance delivery to the immune following mucosal immunization can be enhanced and facilitated system. This strategy was explored in the intranasal delivery of a using adjuvants. Mucosal adjuvants are more important since they subunit influenza HA protein in mice and clinically in healthy target the broad mucosal epithelial cells rather than just the M human subjects where it induced nasal sIgA and systemic anti-HA cells or APCs that represent only a small fraction of the cells at the antibodies137. Liposomes, as discussed earlier, are spherical mucosal epithelia. The most utilized and best characterized nanovesicles consisting of a phospholipid bilayer and cholesterol mucosal adjuvants are the toxoids which are non-toxic derivatives possessing both hydrophobic and hydrophilic layers enclosing an of bacterial toxins originating from the enterotoxigenic E. coli aqueous inner core. This strategy offers an advantage of multiple (ETEC) and Vibrio cholerae, namely heat-labile toxin (LT) and antigen delivery simultaneously since different antigens localize at cholera toxin (CT) respectively, both of which are ATP-ribosylating different portions of the liposome and can be used with different enterotoxins. These toxins have been modified such that they antigens including DNA and RNA platforms as introduced have lost their toxigenicity while still retaining their potent earlier5,101. Liposome-encapsulated DNA vaccine encoding the immunostimulatory properties92. Although these two adjuvants M1 protein of Influenza virus was reported to achieve an efficient have potent immunostimulatory properties and are capable of in-vitro and in vivo expression of the encoded protein. Oral breaking mucosal tolerance to induce mucosal and systemic immunization of mice with this encapsulated DNA vaccine induces responses, their residual toxicity issues have limited their clinical both cellular and humoral responses and augmented interferon- use94. In an attempt to reduce the toxicity of LT, mutation was gamma response. Oral immunization of mice with this vaccine introduced at 2 positions in the A subunit to generate a double also protected from influenza challenge138. ISCOMs are colloidal mutant form (dmLT) which has been shown to be effective and structures synthesized from phospholipids, cholesterol and well tolerated in preclinical and clinical studies144,145. Based on the saponins. They play the dual role of acting as a carrier and as an same approach, CT has also been modified in a novel multiple immunostimulating agent for vaccine delivery. These ISCOMs are mutated form (mmCT) which was shown to be non-toxic and capable of entrapping different antigens and have been shown to induced a protective immunity with cellular and both mucosal and deliver antigen to DCs and facilitate endosomal escape to induce systemic antibodies146. Several other next-generation mucosal T-cell and humoral responses. Intranasal delivery of an influenza adjuvants are currently being explored based on the pattern split virus entrapped in ISCOM induced broad influenza-specific recognition receptor (PRR) ligands since these ligands are widely antibodies which protected against influenza challenge at a expressed by different cells on the mucosal surfaces. Obviously, 10–100 fold lower dose than the split virus alone139. The TLR agonists are mostly researched because of the wide bilosomes are similar to liposomes in structural organization and distribution of TLRs across mucosal epithelial cells. Here, the most adjuvanticity but they are engineered to include bile salts in their promising from preclinical evaluations are the TLR9 and TLR4 formulation. The presence of bile salts offers a better advantage agonists such as unmethylated cytosine-guanine-containing over liposome since liposomes can be disrupted by bile acid in the oligonucleotides (CpG DNA) and monophosphoryl lipid A (MPL) GIT while bilosomes remain stable and are unaffected by bile respectively147–149. Other TLR agonists that have shown results acid5. This strategy is being explored for oral vaccine delivery and include flagellin which is a TLR5 ligand, imiquimoid which is a have been shown to induce both sIgA humoral and cellular TLR7/8 agonist, and poly I:C which is a ligand of TLR 396. In immunity and a balanced Th1/Th2 response following oral addition to the TLR ligands, a promising adjuvant called α-GalCer immunization with influenza subunit protein140. Cell-directed which is a potent activator of the abundant mucosal invariant approaches are also explored to improve antigen uptake and natural killer T cells (iNKT) has been shown in various preclinical vaccine response. This involves strategies targeted towards APC studies to be a good strategy for enhancing mucosal immunity and M-cells on the mucosal surface since these cells are pivotal in with protective cellular and humoral responses150,151. Cytokines driving mucosal and systemic responses. As stated in the previous have also been shown as a possible strategy to enhance mucosal sections, /M-cells are mucosal associated cells which can deliver immunity and improve the efficacy of mucosal vaccines. For mucosal antigens to DC or macrophages by transcytosis. Efforts example, IL-12 co-administration was reported to enhance vaccine are made to target antigen towards the M-cells by utilizing M cell response following intranasal immunization with HIV antigens152. ligands. For example, Ulex europaeus agglutinin 1 (UEA-1) which is More recently, intranasal administration of a recombinant IL-1beta a plant-derived lectin that specifically bind to M cell surface α-L- with Influenza virus HA protein induced a protective immunity fucose residues, has been used as a strategy to target M cells. This evidenced by both HA-specific and strain-specific neutralizing has been used to modify the surface of a PLGA nanoparticle antibodies as well as the local activation of CD103+ CD69+ tissue- delivery system and was shown to induce systemic IgG and resident memory T cells (TRM)153. Recent studies have also mucosal IgA in animal model after oral immunization141. Some reported the induction of mucosal immunity following parenteral bacterial proteins for which receptors are expressed on the M cells immunization and this is mediated via the upregulation of gut- have also been employed in M cell targeting. For example, the homing molecules by parenterally activated immune cells. This GP2 receptor expressed on both murine and human M cell has strategy requires the use of specific adjuvants. For example, it was been shown to bind FimH protein present on E. coli and shown that dmLT, but not CpG, enhanced the upregulation of the Salmonella typhirium, in addition, outer membrane protein H gut-homing α4β7 integrin by antigen-specific T cells following found on Yersinia enterocolitica can bind a receptor on M cells. intradermal immunization and this was driven by CD103+ DCs154. Therefore, recombinant vectors or delivery systems expressing Similarly, subcutaneous administration of retinoic acid with these M cell ligands are being explored for M cells targeting94. A antigen induces the upregulation of α4β7 on both T and B cells similar strategy is applied for mucosal DC targeting. Several DC leading to gut-homing of antigen-specific T cells and IgA+ B cells receptors have been identified and are targeted for vaccine from the regional inguinal lymph node155. delivery, this includes DC-SIGN, CLEC, Langerin, DCIR, Dectin-1. Since DCs are widely distributed along the mucosal surface, including DC ligands in the antigen formulation would enhance CONCLUSION AND FUTURE POSSIBILITIES vaccine uptake and drive vaccine response. Furthermore, some The results from the reviewed studies perhaps provide some basis bacterial species such as Lactobacillus acidophilus have been for the promising potential for mucosal mRNA vaccines for the Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 16 control of infectious diseases. The major challenge is the harsh mRNA vaccines intended for the sublingual, vaginal or rectal mucosal environment and the epithelial barrier, there is a need to immunization as does with the oral and nasal routes. Another develop more efficient delivery systems which ensure the stability possibility is to couple the LNP or any other delivery vehicle to the of the mRNA molecules and improve uptake across the epithelial Fc portion of an antibody whose Fab region is specific for a DC barriers while also minimizing toxicity. The results from previous surface molecule. Indeed, DC targeting could potentially limit off- studies have shown the potential of polymer-based, lipid-based target mRNA vaccine uptake and can be used to preferentially and hybrid delivery vehicles as reviewed above. The lack of target a specific DC lineage which would shape the T cell response experimental studies utilizing the oral mucosa is perhaps due to towards the desired helper phenotype. The mucosal compart- the more challenging nature of the GI environment in terms of mentalization has been shown to impact the tolerogenic potential varying pH and the presence of abundant degradative enzymes. at different segments of the GIT. The upper small intestinal There is a need to develop a delivery vehicle which can overcome segment has been shown to have a higher tolerogenic propensity these challenges. One possibility is by modifying the existing than the distal GI segments156. Hence, developing a delivery delivery systems which have shown success in experimental vehicle that bypasses the upper intestine may be an important models. Future studies can focus on making these delivery strategy. Mucoadhesive substances which increase the residence vehicles safer and more effective, for example, rendering them time of the mRNA molecules have been proven with success in more resistant to degradation in the mucosal environment or experimental models as seen above with cyclodextrin18,127 and improving immunogenicity by the incorporation of adjuvants like other mucoadhesive substances used for vaccine platforms other PRR ligands into the delivery vehicles as seen with the case of than mRNA vaccines such as chitosan and starch157–160. Research mannose-modified LNP which has already been explored with can be focused on incorporating these substances in the delivery mRNA delivery as discussed earlier. Other PRR ligands such as CpG system with particular emphasis on toxicity avoidance. This would oligonucleotide, flagellin and imiquimoid could also be explored increase residence time, a property that is very relevant for nasal in the mRNA delivery vehicle to break the oral tolerance. This also and sublingual immunization to overcome mucociliary action and includes dmLT, mmCT and α-GalCer which have been previously salivary dilution respectively. Since the mucous layer can serve as experimented for mucosal application. These adjuvants could be a barrier to the delivery of substances across mucosal surfaces, expressed on the delivery vehicle or perhaps be alternatively perhaps engineering the delivery system to ensure efficient mRNA-encoded and co-delivered with the antigen-encoding mucus-penetrating activity may improve antigenic mRNA delivery mRNA vaccine candidate. As discussed above, the problem and this can be explored in future studies. Since it has been shown associated with pH variation can be overcome engineering the that gut-homing integrins and retinoic acid could induce homing delivery systems to include bile acids as seen with bileosomes of DC, B cells and T cells to the mucosal surface following which would probably render the carrier vehicle insensitive to the parenteral immunization, it could be possible to direct efforts pH in the GIT. Another possibility is by increasing the dose towards the inclusion of these molecules in the mRNA carrier especially for the oral route such that a high degree of the mRNA molecule or encode these in the mRNA formulation and use this molecules is eventually taken up despite abundant degradative as a combination strategy to mucosal mRNA immunization. Finally, processes, this strategy however may not be economically it would be of immense benefit if mRNA could be engineered and advantageous and may be detrimental to vaccine coverage in a constructed to be resistant by virtue of its inherent design such pandemic scenario. In the context of nasal and oral immunization that it becomes RNAse insensitive and able to withstand the harsh strategies, engineering the mRNA delivery construct to target M mucosal environment. This requires extensive research not only cells at the mucosal surfaces would result in an increased effective regarding the design and delivery but also to monitor the safety of uptake of the mRNA molecules to trigger robust immune response the mRNA vaccines administered mucosally. Although most of the as discussed in the preceding section and this can be explored in strategies we proposed have been explored for other vaccine future studies as well. Perhaps, the mRNA delivery vehicle could platforms, as it stands now, most of these strategies are rather be designed to include M cell ligands such as UEA-1 or microbial speculative in the context of mRNA vaccine delivery. Nonetheless, ligand FimH which would preferentially enhance uptake by M we believe that research efforts to exploit the proposed strategies cells. Another possibility is to use an antibody-guided targeting would accelerate and facilitate effective utilization of mucosal where the delivery vehicle is coupled to the Fc portion of an delivery to attain the full potential of mRNA vaccine in the fight antibody whose Fab region is specific for an M cell surface against infectious diseases. molecule. One important challenge with mucosal vaccination particularly through the oral and sublingual routes is the Reporting summary tolerability potential as highlighted earlier; inclusion and co- Further information on research design is available in the Nature delivery of cytokine or chemokine-encoding mRNA with the Research Reporting Summary linked to this article. antigen-encoding mRNA may be a potential strategy of breaking this tolerance and triggering immunogenicity, in addition to PRR ligands. Since different cellular phenotype are required for Received: 8 September 2021; Accepted: 13 May 2022; different class of pathogens (e.g virus, bacteria and helminth), the specific cytokines that imprint the desired T cell response could possibly be mRNA-encoded and co-delivered with the antigen-encoding mRNA. For example, including IFN-γ for TH1/ REFERENCES CD8 response or IL-17 for TH17 response against viruses and 1. Wang, L. & Crameri, G. Emerging zoonotic viral diseases. Rev. Sci. Tech. 33, extracellular bacteria/fungi respectively. This strategy can be 569–581 (2014). combined with a cell-targeted delivery to induce a significant 2. Marston, H. D., Folkers, G. K., Morens, D. M. & Fauci, A. S. Emerging viral diseases: response that breaks tolerance, but care should be taken to avoid confronting threats with new technologies. Sci. Transl. Med. 6, detrimental inflammatory response. Since DCs are distributed all 253ps210–253ps210 (2014). over the mucosal compartments, targeting these cells is indeed a 3. Ogbu, O., Ajuluchukwu, E. & Uneke, C. Lassa fever in West African sub-region: an promising strategy for vaginal and rectal mucosa which are devoid overview. J. Vector Borne Dis. 44, 1 (2007). of M cells or organised lymphoid tissues and of course, other 4. Hartman, A. L., Towner, J. S. & Nichol, S. T. Ebola and marburg hemorrhagic mucosal compartments. Numerous DC ligands have been fever. Clin. Lab. Med. 30, 161–177 (2010). employed in the context of DC targeting such as DC-SIGN, DCIR 5. Ramirez, J. E. V., Sharpe, L. A. & Peppas, N. A. Current state and challenges in developing oral vaccines. Adv. Drug Deliv. Rev. 114, 116–131 (2017). and CLEC-9a, these can be included in the delivery vehicle of npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 17 6. Torrieri-Dramard, L. et al. Intranasal DNA vaccination induces potent mucosal 38. Debertin, A. et al. Coincidence of different structures of mucosa‐associated and systemic immune responses and cross-protective immunity against influ- lymphoid tissue (MALT) in the respiratory tract of children: no indications for enza viruses. Mol. Ther. 19, 602–611 (2011). enhanced mucosal immunostimulation in sudden infant death syndrome (SIDS). 7. Munster, V. J., Koopmans, M., van Doremalen, N., van Riel, D. & de Wit, E. A novel Clin. Exp. Immunol. 146, 54–59 (2006). coronavirus emerging in China—key questions for impact assessment. N. Engl. J. 39. Debertin, A. et al. Nasal‐associated lymphoid tissue (NALT): frequency and Med. 382, 692–694 (2020). localization in young children. Clin. Exp. Immunol. 134, 503–507 (2003). 8. Organization, W. H. WHO Coronavirus dashboard, https://covid19.who.int/ 40. Takaki, H. et al. Toll-like receptor 3 in nasal CD103+ dendritic cells is involved in (2021). immunoglobulin A production. Mucosal Immunol. 11, 82–96 (2018). 9. Dickinson, M. Food frights: COVID-19 and the specter of hunger. Agriculture 41. Nacer, A. et al. Imaging murine NALT following intranasal immunization with Hum. Values 37, 589–590 (2020). flagellin-modified circumsporozoite protein malaria vaccines. Mucosal Immunol. 10. Loomis, R. J. & Johnson, P. R. Emerging vaccine technologies. Vaccines 3, 7, 304–314 (2014). 429–447 (2015). 42. Rochereau, N. et al. Delivery of antigen to nasal-associated lymphoid tissue 11. Greenwood, B. The contribution of vaccination to global health: past, present microfold cells through secretory IgA targeting local dendritic cells confers and future. Philos. Trans. R. Soc. B: Biol. Sci. 369, 20130433 (2014). protective immunity. J. Allergy Clin. Immunol. 137, 214–222. e212 (2016). 12. BashirBello, M., Peeters, B. P. & RahmanOmar, A. Diagnostic and vaccination 43. Kato, A., Truong-Tran, A. Q., Scott, A. L., Matsumoto, K. & Schleimer, R. P. Airway approaches for newcastle disease virus in poultry: the current and emerging epithelial cells produce B cell-activating factor of TNF family by an IFN- perspectives. Viruses 12, 451 (2018). β-dependent mechanism. J. Immunol. 177, 7164–7172 (2006). 13. Bello, M. B. et al. Exploring the prospects of engineered Newcastle disease virus 44. Lapuente, D. et al. Protective mucosal immunity against SARS-CoV-2 after het- in modern vaccinology. Viruses 12, 451 (2020). erologous systemic prime-mucosal boost immunization. Nat. Commun. 12, 1–14 14. Schlake, T., Thess, A., Fotin-Mleczek, M. & Kallen, K.-J. Developing mRNA-vaccine (2021). technologies. RNA Biol. 9, 1319–1330 (2012). 45. Presland, R. B. & Dale, B. A. Epithelial structural proteins of the skin and oral 15. Jackson, N., Kester, K., Casimiro, D., Gurunathan, S. & DeRosa, F. The promise cavity: function in health and disease. Crit. Rev. Oral. Biol. Med. 11, 383–408 of mRNA vaccines: a biotech and industrial perspective. npj Vaccines 5, 1–6 (2000). (2020). 46. Teubl, B. J. et al. The oral cavity as a biological barrier system: design of an 16. Ulmer, J. B. & Geall, A. J. Recent innovations in mRNA vaccines. Curr. Opin. advanced buccal in vitro permeability model. Eur. J. Pharmaceutics Biopharma- Immunol. 41, 18–22 (2016). ceutics 84, 386–393 (2013). 17. Karikó, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition 47. Kraan, H. et al. Buccal and sublingual vaccine delivery. J. controlled release 190, by toll-like receptors: the impact of nucleoside modification and the evolu- 580–592 (2014). tionary origin of RNA. Immunity 23, 165–175 (2005). 48. Paris, A., Colomb, E., Verrier, B., Anjuère, F. & Monge, C. Sublingual vaccination 18. Li, M. et al. Enhanced intranasal delivery of mRNA vaccine by overcoming the and delivery systems. J. Controlled Release 332, 553–562 (2021). nasal epithelial barrier via intra-and paracellular pathways. J. Controlled Release 49. Kumamoto, Y. & Iwasaki, A. Unique features of antiviral immune system of the 228, 9–19 (2016). vaginal mucosa. Curr. Opin. Immunol. 24, 411–416 (2012). 19. Zhang, N., Van Crombruggen, K., Gevaert, E. & Bachert, C. Barrier function of the 50. Iwasaki, A. Antiviral immune responses in the genital tract: clues for vaccines. nasal mucosa in health and type‐2 biased airway diseases. Allergy 71, 295–307 Nat. Rev. Immunol. 10, 699–711 (2010). (2016). 51. Zhang, N.-N. et al. A thermostable mRNA vaccine against COVID-19. Cell 182, 20. Ahluwalia, B., Magnusson, M. K. & Öhman, L. Mucosal immune system of the 1271–1283.e1216 (2020). gastrointestinal tract: maintaining balance between the good and the bad. 52. Tan, L. & Sun, X. Recent advances in mRNA vaccine delivery. Nano Res. 11, Scand. J. Gastroenterol. 52, 1185–1193 (2017). 5338–5354 (2018). 21. Allaire, J. M. et al. The intestinal epithelium: central coordinator of mucosal 53. Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can immunity. Trends Immunol. 39, 677–696 (2018). extend translation duration in vivo. Mol. Cell 74, 508–520. e504 (2019). 22. Stephens, P. & Genever, P. Non‐epithelial oral mucosal progenitor cell popula- 54. Ahammad, I. & Lira, S. S. Designing a novel mRNA vaccine against SARS-CoV-2: tions. Oral. Dis. 13, 1–10 (2007). An immunoinformatics approach. Int. J. Biol. Macromol. 162, 820–837 (2020). 23. Lechanteur, A., das Neves, J. & Sarmento, B. The role of mucus in cell-based 55. Maruggi, G., Zhang, C., Li, J., Ulmer, J. B. & Yu, D. mRNA as a transformative models used to screen mucosal drug delivery. Adv. Drug Deliv. Rev. 124, 50–63 technology for vaccine development to control infectious diseases. Mol. Ther. (2018). 27, 757–772 (2019). 24. Cesta, M. F. Normal structure, function, and histology of mucosa-associated 56. Sharifnia, Z., Bandehpour, M., Kazemi, B. & Zarghami, N. Design and develop- lymphoid tissue. Toxicol. Pathol. 34, 599–608 (2006). ment of modified mRNA encoding core antigen of hepatitis C virus: a possible 25. Lin, M., Du, L., Brandtzaeg, P. & Pan-Hammarström, Q. IgA subclass switch application in vaccine production. Iran. Biomed. J. 23, 57 (2019). recombination in human mucosal and systemic immune compartments. 57. Kramps, T. & Probst, J. Messenger RNA‐based vaccines: progress, challenges, Mucosal Immunol. 7, 511–520 (2014). applications. Wiley Interdiscip. Rev.: RNA 4, 737–749 (2013). 26. Brandtzaeg, P. Function of mucosa-associated lymphoid tissue in antibody 58. Zeng, C. et al. Leveraging mRNAs sequences to express SARS-CoV-2 antigens formation. Immunological Investig. 39, 303–355 (2010). in vivo. BioRxiv (2020). 27. Gutzeit, C., Magri, G. & Cerutti, A. Intestinal IgA production and its role in host‐ 59. Zhang, C., Maruggi, G., Shan, H. & Li, J. Advances in mRNA vaccines for infectious microbe interaction. Immunological Rev. 260, 76–85 (2014). diseases. Front. Immunol. 10, 594 (2019). 28. Sakai, Y. & Kobayashi, M. Lymphocyte ‘homing’and chronic inflammation. 60. Anderson, B. R. et al. Incorporation of pseudouridine into mRNA enhances Pathol. Int. 65, 344–354 (2015). translation by diminishing PKR activation. Nucleic Acids Res. 38, 5884–5892 29. Schroeder, H. W. Jr & Cavacini, L. Structure and function of immunoglobulins. J. (2010). Allergy Clin. Immunol. 125, S41–S52 (2010). 61. Andries, O. et al. N1-methylpseudouridine-incorporated mRNA outperforms 30. McGuckin, M. A., Lindén, S. K., Sutton, P. & Florin, T. H. Mucin dynamics and pseudouridine-incorporated mRNA by providing enhanced protein expression enteric pathogens. Nat. Rev. Microbiol. 9, 265–278 (2011). and reduced immunogenicity in mammalian cell lines and mice. J. Controlled 31. Fujihashi, K. et al. Peyer’s patches are required for oral tolerance to proteins. Release 217, 337–344 (2015). Proc. Natl Acad. Sci. USA 98, 3310–3315 (2001). 62. Frederickson, R. & Herzog, R. W. RNA-based vaccines and innate immune acti- 32. Panneerselvam, D. & Budh, D. P. Peyer Patches. StatPearls [Internet] https:// vation: Not too hot and not too cold. Mol. Therapy 29, 1365–1366 (2021). europepmc.org/article/MED/32491389 (2021). 63. Knezevic, I., Liu, M. A., Peden, K., Zhou, T. & Kang, H.-N. Development of mRNA. 33. Lycke, N. Recent progress in mucosal vaccine development: potential and lim- Vaccines: Sci. Regulatory Issues Vaccines 9, 81 (2021). itations. Nat. Rev. Immunol. 12, 592–605 (2012). 64. Igyártó, B. Z., Jacobsen, S. & Ndeupen, S. Future considerations for the mRNA- 34. Provine, N. M. & Klenerman, P. MAIT cells in health and disease. Annu. Rev. lipid nanoparticle vaccine platform. Curr. Opin. Virol. 48, 65–72 (2021). Immunol. 38, 203–228 (2020). 65. Pepini, T. et al. Induction of an IFN-mediated antiviral response by a self- 35. McDole, J. R. et al. Goblet cells deliver luminal antigen to CD103+ dendritic cells amplifying RNA vaccine: implications for vaccine design. J. Immunol. 198, in the small intestine. Nature 483, 345–349 (2012). 4012–4024 (2017). 36. Regoli, M., Bertelli, E., Gulisano, M. & Nicoletti, C. The multifaceted personality of 66. Verbeke, R., Lentacker, I., De Smedt, S. C. & Dewitte, H. Three decades of mes- intestinal CX3CR1+ macrophages. Trends Immunol. 38, 879–887 (2017). senger RNA vaccine development. Nano Today 28, 100766 (2019). 37. Janeway Jr, C. A., Travers, P., Walport, M. & Shlomchik, M. J. Principles of innate 67. Devoldere, J., Dewitte, H., De Smedt, S. C. & Remaut, K. Evading innate immunity and adaptive immunity. In Immunobiology: The Immune System in Health and in nonviral mRNA delivery: don’t shoot the messenger. Drug Discov. today 21, Disease. 5th edn. Garland Science (2001). 11–25 (2016). Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 18 68. Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines—a new era 97. Foged, C. Subunit vaccines of the future: the need for safe, customized and in vaccinology. Nat. Rev. Drug Discov. 17, 261 (2018). optimized particulate delivery systems. Therapeutic Deliv. 2, 1057–1077 (2011). 69. Zhong, Z. et al. mRNA therapeutics deliver a hopeful message. Nano Today 23, 98. Kantele, A. et al. Differences in immune responses induced by oral and rectal 16–39 (2018). immunizations with Salmonella typhi Ty21a: evidence for compartmentalization 70. Jackson, N. A., Kester, K. E., Casimiro, D., Gurunathan, S. & DeRosa, F. The promise of within the common mucosal immune system in humans. Infect. Immun. 66, mRNA vaccines: a biotech and industrial perspective. npj Vaccines 5, 1–6 (2020). 5630–5635 (1998). 71. Beissert, T. et al. Improvement of in vivo expression of genes delivered by self- 99. Leigh, S. A., Evans, J. D., Collier, S. D. & Branton, S. L. The impact of vaccination amplifying RNA using vaccinia virus immune evasion proteins. Hum. Gene Ther. route on Mycoplasma gallisepticum vaccine efficacy. Poult. Sci. 97, 3072–3075 28, 1138–1146 (2017). (2018). 72. Liu, Y., Chin, J. M., Choo, E. L. & Phua, K. K. Messenger RNA translation 100. Kiyono, H. & Fukuyama, S. NALT-versus Peyer’s-patch-mediated mucosal enhancement by immune evasion proteins: a comparative study between EKB immunity. Nat. Rev. Immunol. 4, 699–710 (2004). (vaccinia virus) and NS1 (influenza A virus). Sci. Rep. 9, 1–9 (2019). 101. Jabbal-Gill, I. Nasal vaccine innovation. J. drug Target. 18, 771–786 (2010). 73. Blakney, A. K. et al. Innate Inhibiting Proteins Enhance Expression and Immu- 102. Pedersen, G. & Cox, R. The mucosal vaccine quandary: intranasal vs. sublingual nogenicity of Self-Amplifying RNA. Mol. Therapy 29, 1174–1185 (2020). immunization against influenza. Hum. Vaccines Immunotherapeutics 8, 689–693 74. Pardi, N. et al. Expression kinetics of nucleoside-modified mRNA delivered in (2012). lipid nanoparticles to mice by various routes. J. Controlled Release 217, 345–351 103. Carter, N. J. & Curran, M. P. Live attenuated influenza vaccine (FluMist®; Fluenz™). (2015). Drugs 71, 1591–1622 (2011). 75. Bozzuto, G. & Molinari, A. Liposomes as nanomedical devices. Int. J. Nanomed. 104. Tribble, D. et al. Safety and immunogenicity of a Shigella flexneri 2a Invaplex 50 10, 975 (2015). intranasal vaccine in adult volunteers. Vaccine 28, 6076–6085 (2010). 76. Zhi, D. et al. The headgroup evolution of cationic lipids for gene delivery. Bio- 105. Bomsel, M. et al. Immunization with HIV-1 gp41 subunit virosomes induces conjugate Chem. 24, 487–519 (2013). mucosal antibodies protecting nonhuman primates against vaginal SHIV chal- 77. Guan, S. & Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics lenges. Immunity 34, 269–280 (2011). using nonviral vector-based delivery systems. Gene Ther. 24, 133–143 (2017). 106. Marks, E., Helgeby, A., Andersson, J. O., Schön, K. & Lycke, N. Y. CD4+ T‐cell 78. Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for ther- immunity in the female genital tract is critically dependent on local mucosal apeutic mRNA delivery. Nat. Rev. Mater. 2, 1–17 (2017). immunization. Eur. J. Immunol. 41, 2642–2653 (2011). 79. Linares-Fernández, S., Lacroix, C., Exposito, J.-Y. & Verrier, B. Tailoring mRNA 107. Lewis, D. J. et al. Phase I randomised clinical trial of an HIV-1CN54, clade C, vaccine to balance innate/adaptive immune response. Trends Mol. Med. 26, trimeric envelope vaccine candidate delivered vaginally. PLoS ONE 6, e25165 311–323 (2020). (2011). 80. Granot, Y. & Peer, D. Delivering the right message: Challenges and opportunities 108. Belyakov, I. M. et al. Impact of vaccine-induced mucosal high-avidity CD8+ in lipid nanoparticles-mediated modified mRNA therapeutics—An innate CTLs in delay of AIDS viral dissemination from mucosa. Blood 107, 3258–3264 immune system standpoint. In Seminars in Immunology 34, 68–77, Academic (2006). Press (2017). 109. Belyakov, I. M. et al. Mucosal AIDS vaccine reduces disease and viral load in gut 81. van Riel, D. & de Wit, E. Next-generation vaccine platforms for COVID-19. Nat. reservoir and blood after mucosal infection of macaques. Nat. Med. 7, Mater. 19, 810–812 (2020). 1320–1326 (2001). 82. Lombardi, A. et al. MINI REVIEW Immunological consequences of immunization 110. Domm, W. et al. Robust antigen-specific humoral immune responses to sub- with COVID-19 mRNA vaccines: Preliminary results. Front. Immunol. 12, 677 lingually delivered adenoviral vectors encoding HIV-1 Env: association with (2021). mucoadhesion and efficient penetration of the sublingual barrier. Vaccine 29, 83. Park, J. W., Lagniton, P. N., Liu, Y. & Xu, R.-H. mRNA vaccines for COVID-19: what, 7080–7089 (2011). why and how. Int. J. Biol. Sci. 17, 1446 (2021). 111. Cho, H.-J. et al. Enhanced humoral and cellular immune responses after sub- 84. Meo, S., Bukhari, I., Akram, J., Meo, A. & Klonoff, D. COVID-19 vaccines: com- lingual immunization against human papillomavirus 16 L1 protein with adju- parison of biological, pharmacological characteristics and adverse effects of vants. Vaccine 28, 2598–2606 (2010). Pfizer/BioNTech and Moderna Vaccines. Eur. Rev. Med. Pharmacol. Sci. 25, 112. Czerkinsky, C., Çuburu, N., Kweon, M.-N., Anjuere, F. & Holmgren, J. Sublingual 1663–1669 (2021). vaccination. Hum. vaccines 7, 110–114 (2011). 85. Anderson, E. J. et al. Safety and immunogenicity of SARS-CoV-2 mRNA-1273 113. Song, J.-H. et al. Sublingual vaccination with influenza virus protects mice vaccine in older adults. N. Engl. J. Med. 383, 2427–2438 (2020). against lethal viral infection. Proc. Natl Acad. Sci. USA 105, 1644–1649 (2008). 86. Bajema, K. L. et al. Effectiveness of COVID-19 mRNA vaccines against COVID- 114. Flach, C.-F. et al. A truncated form of HpaA is a promising antigen for use in a 19–associated hospitalization—five veterans affairs medical centers, United vaccine against Helicobacter pylori. Vaccine 29, 1235–1241 (2011). States, February 1–August 6, 2021. Morbidity Mortal. Wkly. Rep. 70, 1294 (2021). 115. Echchannaoui, H. et al. Intravaginal immunization of mice with recombinant 87. (CDC), C. f. D. C. Pfizer-BioNTech COVID-19 Vaccine, Storage and Handling Summary, Salmonella enterica serovar Typhimurium expressing human papillomavirus https://www.cdc.gov/vaccines/covid-19/info-by-product/pfizer/downloads/storage- type 16 antigens as a potential route of vaccination against cervical cancer. summary.pdf (2021). Infect. Immun. 76, 1940–1951 (2008). 88. Self, W. H. et al. Comparative effectiveness of Moderna, Pfizer-BioNTech, and 116. Domingos-Pereira, S. et al. Intravaginal TLR agonists increase local vaccine- Janssen (Johnson & Johnson) vaccines in preventing COVID-19 hospitalizations specific CD8 T cells and human papillomavirus-associated genital-tumor among adults without immunocompromising conditions—United States, regression in mice. Mucosal Immunol. 6, 393–404 (2013). March–August 2021. Morbidity Mortal. Wkly. Rep. 70, 1337 (2021). 117. Seavey, M. M. & Mosmann, T. R. Estradiol-induced vaginal mucus inhibits anti- 89. Tenforde, M. W. et al. Sustained effectiveness of Pfizer-BioNTech and Moderna gen penetration and CD8+ T cell priming in response to intravaginal immuni- vaccines against COVID-19 associated hospitalizations among adults—United zation. Vaccine 27, 2342–2349 (2009). States, March–July 2021. Morbidity Mortal. Wkly. Rep. 70, 1156 (2021). 118. Coffey, J. W., Gaiha, G. D. & Traverso, G. Oral biologic delivery: advances toward 90. Chahal, J. S. et al. Dendrimer-RNA nanoparticles generate protective immunity oral subunit, DNA, and mRNA vaccines and the potential for mass vaccination against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a during pandemics. Annu. Rev. Pharmacol. Toxicol. 61, 517 (2021). single dose. Proc. Natl Acad. Sci. USA 113, E4133–E4142 (2016). 119. Tucker, S. N., Tingley, D. W. & Scallan, C. D. Oral adenoviral-based vaccines: 91. Cagigi, A. & Loré, K. Immune responses induced by MRNA vaccination in mice, historical perspective and future opportunity. Expert Rev. Vaccines 7, 25–31 monkeys and humans. Vaccines 9, 61 (2021). (2008). 92. Coffey, J. W., Gaiha, G. D. & Traverso, G. Oral biologic delivery: advances toward 120. Pasetti, M. F., Simon, J. K., Sztein, M. B. & Levine, M. M. Immunology of gut oral subunit, DNA, and mRNA vaccines and the potential for mass vaccination mucosal vaccines. Immunol. Rev. 239, 125–148 (2011). during pandemics. Annu. Rev. Pharmacol. Toxicol. 61, 517–540 (2021). 121. Kirkpatrick, B. et al. Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC- 93. Miquel‐Clopés, A., Bentley, E., Stewart, J. & Carding, S. Mucosal vaccines and ssaV-) M01ZH09, with a defined mutation in the Salmonella pathogenicity island technology. Clin. Exp. Immunol. 196, 205–214 (2019). 2, as a live, oral typhoid vaccine in human volunteers. Vaccine 24, 116–123 94. Li, M. et al. Mucosal vaccines: strategies and challenges. Immunol. Lett. 217, (2006). 116–125 (2020). 122. Kim, S.-H. & Jang, Y.-S. The development of mucosal vaccines for both mucosal 95. Lavelle, E. C. & Ward, R. W. Mucosal vaccines—fortifying the frontiers. Nat. Rev. and systemic immune induction and the roles played by adjuvants. Clin. Exp. Immunol. 22, 236–250 (2022). Vaccin. Res. 6, 15–21 (2017). 96. Nizard, M. et al. Mucosal vaccines: novel strategies and applications for the 123. Langridge, W., Dénes, B. & Fodor, I. Cholera toxin B subunit modulation of control of pathogens and tumors at mucosal sites. Hum. Vaccines Immu- mucosal vaccines for infectious and autoimmune diseases. Curr. Opin. Investig. notherapeutics 10, 2175–2187 (2014). Drugs 11, 919–928 (2010). npj Vaccines (2022) 71 Published in partnership with the Sealy Institute for Vaccine Sciences S.A. Hameed et al. 19 124. Ambrose, C. S., Wu, X., Jones, T. & Mallory, R. M. The role of nasal IgA in children 150. Longet, S. et al. An oral alpha-galactosylceramide adjuvanted Helicobacter vaccinated with live attenuated influenza vaccine. Vaccine 30, 6794–6801 pylori vaccine induces protective IL-1R-and IL-17R-dependent Th1 responses. (2012). NPJ Vaccines 4, 1–10 (2019). 125. Zeng, C., Zhang, C., Walker, P. G. & Dong, Y. Formulation and delivery tech- 151. Davitt, C. J. et al. Alpha-galactosylceramide enhances mucosal immunity to oral nologies for mRNA vaccines. In Current Topics in Microbiology and Immunology whole-cell cholera vaccines. Mucosal Immunol. 12, 1055–1064 (2019). 217, 1–40 (2020). 152. Albu, D. I. et al. Intranasal vaccination using interleukin-12 and cholera toxin 126. Lorenzi, J. C. et al. Intranasal vaccination with messenger RNA as a new subunit B as adjuvants to enhance mucosal and systemic immunity to human approach in gene therapy: use against tuberculosis. BMC Biotechnol. 10, 1–11 immunodeficiency virus type 1 glycoproteins. J. Virol. 77, 5589–5597 (2003). (2010). 153. Lapuente, D. et al. IL-1β as mucosal vaccine adjuvant: the specific induction of 127. Li, M. et al. Engineering intranasal mRNA vaccines to enhance lymph node tissue-resident memory T cells improves the heterosubtypic immunity against trafficking and immune responses. Acta biomaterialia 64, 237–248 (2017). influenza A viruses. Mucosal Immunol. 11, 1265–1278 (2018). 128. Zhuang, X. et al. mRNA vaccines encoding the HA protein of influenza A H1N1 154. Frederick, D. R. et al. Adjuvant selection regulates gut migration and phenotypic virus delivered by cationic lipid nanoparticles induce protective immune diversity of antigen-specific CD4+ T cells following parenteral immunization. responses in mice. Vaccines 8, 123 (2020). Mucosal Immunol. 11, 549–561 (2018). 129. Tiwari, P. M. et al. Engineered mRNA-expressed antibodies prevent respiratory 155. Hammerschmidt, S. I. et al. Retinoic acid induces homing of protective T and B syncytial virus infection. Nat. Commun. 9, 1–15 (2018). cells to the gut after subcutaneous immunization in mice. J. Clin. Investig. 121, 130. Lindsay, K. E. et al. Aerosol delivery of synthetic mRNA to vaginal mucosa leads 3051–3061 (2011). to durable expression of broadly neutralizing antibodies against HIV. Mol. Ther. 156. Esterházy, D. et al. Compartmentalized gut lymph node drainage dictates 28, 805–819 (2020). adaptive immune responses. Nature 569, 126–130 (2019). 131. Phua, K. K., Staats, H. F., Leong, K. W. & Nair, S. K. Intranasal mRNA nanoparticle 157. Park, J. S., Oh, Y. K., Kang, M. J. & Kim, C. K. Enhanced mucosal and systemic vaccination induces prophylactic and therapeutic anti-tumor immunity. Sci. Rep. immune responses following intravaginal immunization with human papillo- 4, 1–7 (2014). mavirus 16 L1 virus‐like particle vaccine in thermosensitive mucoadhesive 132. Mai, Y. et al. Intranasal delivery of cationic liposome-protamine complex mRNA delivery systems. J. Med. Virol. 70, 633–641 (2003). vaccine elicits effective anti-tumor immunity. Cell. Immunol. 354, 104143 (2020). 158. Spinner, J. L. et al. Methylglycol chitosan and a synthetic TLR4 agonist enhance 133. Garg, N. K., Mangal, S., Khambete, H., Sharma, P. K. & Tyagi, R. K. Mucosal delivery immune responses to influenza vaccine administered sublingually. Vaccine 33, of vaccines: role of mucoadhesive/biodegradable polymers. Recent Pat. Drug 5845–5853 (2015). Deliv. formulation 4, 114–128 (2010). 159. Fan, Y., Sahdev, P., Ochyl, L. J., Akerberg, J. J. & Moon, J. J. Cationic 134. Jabbal-Gill, I., Watts, P. & Smith, A. Chitosan-based delivery systems for mucosal liposome–hyaluronic acid hybrid nanoparticles for intranasal vaccination with vaccines. Expert Opin. drug Deliv. 9, 1051–1067 (2012). subunit antigens. J. controlled release 208, 121–129 (2015). 135. Lin, C.-Y. et al. Biodegradable polymeric microsphere-based vaccines and their 160. Li, H.-S. et al. Nasal immunization with mannan-decorated mucoadhesive applications in infectious diseases. Hum. Vaccines Immunotherapeutics 11, HPMCP microspheres containing ApxIIA toxin induces protective immunity 650–656 (2015). against challenge infection with Actinobacillus pleuropneumoiae in mice. J. 136. Serradell, M. C. et al. Efficient oral vaccination by bioengineering virus-like Controlled Release 233, 114–125 (2016). particles with protozoan surface proteins. Nat. Commun. 10, 1–15 (2019). 161. Huang, Q., Zeng, J. & Yan, J. COVID-19 mRNA vaccines. J. Genet. Genomics 48, 137. Treanor, J. et al. Intranasal administration of a proteosome-influenza vaccine is 107–114 (2021). well-tolerated and induces serum and nasal secretion influenza antibodies in 162. John, S. et al. Multi-antigenic human cytomegalovirus mRNA vaccines that elicit healthy human subjects. Vaccine 24, 254–262 (2006). potent humoral and cell-mediated immunity. Vaccine 36, 1689–1699 (2018). 138. Liu, J. et al. Oral vaccination with a liposome‐encapsulated influenza DNA 163. Alberer, M. et al. Safety and immunogenicity of a mRNA rabies vaccine in vaccine protects mice against respiratory challenge infection. J. Med. Virol. 86, healthy adults: an open-label, non-randomised, prospective, first-in-human 886–894 (2014). phase 1 clinical trial. Lancet 390, 1511–1520 (2017). 139. Sanders, M. T., Deliyannis, G., Pearse, M. J., McNamara, M. K. & Brown, L. E. Single 164. Meyer, M. et al. Modified mRNA-based vaccines elicit robust immune responses dose intranasal immunization with ISCOMATRIX™ vaccines to elicit antibody- and protect guinea pigs from Ebola virus disease. J. Infect. Dis. 217, 451–455 (2018). mediated clearance of influenza virus requires delivery to the lower respiratory 165. Richner, J. M. et al. Vaccine mediated protection against Zika virus-induced tract. Vaccine 27, 2475–2482 (2009). congenital disease. Cell 170, 273–283.e212 (2017). 140. Conacher, M., Alexander, J. & Brewer, J. M. Oral immunisation with peptide and 166. Feldman, R. A. et al. mRNA vaccines against H10N8 and H7N9 influenza viruses protein antigens by formulation in lipid vesicles incorporating bile salts (bilo- of pandemic potential are immunogenic and well tolerated in healthy adults in somes). Vaccine 19, 2965–2974 (2001). phase 1 randomized clinical trials. Vaccine 37, 3326–3334 (2019). 141. Du, L. et al. Targeted delivery of GP5 antigen of PRRSV to M cells enhances the 167. Allard, S. D. et al. A phase I/IIa immunotherapy trial of HIV-1-infected patients antigen-specific systemic and mucosal immune responses. Front. Cell. Infect. with Tat, Rev and Nef expressing dendritic cells followed by treatment inter- Microbiol. 8, 7 (2018). ruption. Clin. Immunol. 142, 252–268 (2012). 142. Blank, F., Stumbles, P. & von Garnier, C. Opportunities and challenges of the 168. Espeseth, A. S. et al. Modified mRNA/lipid nanoparticle-based vaccines expres- pulmonary route for vaccination. Expert Opin. Drug Deliv. 8, 547–563 (2011). sing respiratory syncytial virus F protein variants are immunogenic and pro- 143. Mohamadzadeh, M. A., Duong, T., Sandwick, S., Hoover, T. & Klaenhammer, T. tective in rodent models of RSV infection. NPJ Vaccines 5, 1–14 (2020). Dendritic cell targeting of Bacillus anthracis protective antigen expressed by 169. Wollner, C. J. et al. A dengue virus serotype 1 mRNA-LNP vaccine elicits pro- Lactobacillus acidophilus protects mice from lethal challenge. Proc. Natl Acad. tective immune responses. J. Virol. 95, e02482–02420 (2021). Sci. USA 106, 4331–4336 (2009). 170. Shaw, C. et al. Safety and immunogenicity of a mRNA-based chikungunya 144. El-Kamary, S. S. et al. Safety and immunogenicity of a single oral dose of vaccine in a phase 1 dose-ranging trial. Int. J. Infect. Dis. 79, 17 (2019). recombinant double mutant heat-labile toxin derived from enterotoxigenic 171. Goyal, M. et al. Recent development in the strategies projected for chikungunya Escherichia coli. Clin. Vaccin. Immunol. 20, 1764–1770 (2013). vaccine in humans. Drug Des., Dev. Ther. 12, 4195 (2018). 145. Summerton, N. A. et al. Toward the development of a stable, freeze-dried for- 172. Saluja, T., Mogasale, V. V., Excler, J.-L., Kim, J. H. & Mogasale, V. An overview of mulation of Helicobacter pylori killed whole cell vaccine adjuvanted with a VaxchoraTM, a live attenuated oral cholera vaccine. Hum. Vaccines immu- novel mutant of Escherichia coli heat-labile toxin. Vaccine 28, 1404–1411 (2010). notherapeutics 16, 42–50 (2020). 146. Holmgren, J. et al. Preclinical immunogenicity and protective efficacy of an oral 173. Collins, N. D. et al. Live oral adenovirus type 4 and type 7 vaccine induces Helicobacter pylori inactivated whole cell vaccine and multiple mutant cholera durable antibody response. Vaccines 8, 411 (2020). toxin: a novel and non-toxic mucosal adjuvant. Vaccine 36, 6223–6230 (2018). 147. Fernandez, S., Cisney, E. D. & Ulrich, R. G. Enhancement of serum and mucosal immune responses to a Haemophilus influenzae Type B vaccine by intranasal delivery. Clin. Vaccin. Immunol. 20, 1690–1696 (2013). ACKNOWLEDGEMENTS 148. Huang, C. F. et al. Effect of neonatal sublingual vaccination with native or S.A.H. and G.K.D. are registered in the EMJMD LIVE (Erasmus + Mundus Joint Master denatured ovalbumin and adjuvant CpG or cholera toxin on systemic and Degree Leading International Vaccinology Education), co-funded by the EACEA mucosal immunity in mice. Scand. J. Immunol. 68, 502–510 (2008). (Education, Audiovisual and Culture Executive Agency, award 2018–1484) of the 149. Pesce, I. et al. Intranasal administration of CpG induces a rapid and transient European Commission. S.A.H. receives a scholarship from the EACEA and G.K.D. cytokine response followed by dendritic and natural killer cell activation and receives a scholarship from the Department of Science and Technology-Science recruitment in the mouse lung. J. Innate Immun. 2, 144–159 (2010). Education Institute (DOST-SEI) of the Philippines to study in the EMJMD LIVE. Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2022) 71 S.A. Hameed et al. 20 AUTHOR CONTRIBUTIONS Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims S.A.H.: Conceptualization, Writing—original draft, Writing—review and editing. S.P.: in published maps and institutional affiliations. Writing—review and editing. D.J.: Writing- review and editing. G.K.D.: Writing- review and editing. B.B.T.: Writing—original draft, Writing—review and editing. 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