Commensal microbiome and gastrointestinal mucosal immunity: Harmony and conflict with our closest neighbor.

BACKGROUND: The gastrointestinal tract contains a wide range of microorganisms that have evolved alongside the immune system of the host. The intestinal mucosa maintains balance within the intestines by utilizing the mucosal immune system, which is controlled by the complex gut mucosal immune network. OBJECTIVE: This review aims to comprehensively introduce current knowledge of the gut mucosal immune system, focusing on its interaction with commensal bacteria. RESULTS: The gut mucosal immune network includes gut-associated lymphoid tissue, mucosal immune cells, cytokines, and chemokines. The connection between microbiota and the immune system occurs through the engagement of bacterial components with pattern recognition receptors found in the intestinal epithelium and antigen-presenting cells. This interaction leads to the activation of both innate and adaptive immune responses. The interaction between the microbial community and the host is vital for maintaining the balance and health of the host's mucosal system. CONCLUSION: The gut mucosal immune network maintains a delicate equilibrium between active immunity, which defends against infections and damaging non-self antigens, and immunological tolerance, which allows for the presence of commensal microbiota and dietary antigens. This balance is crucial for the maintenance of intestinal health and homeostasis. Disturbance of gut homeostasis leads to enduring or severe gastrointestinal ailments, such as colorectal cancer and inflammatory bowel disease. Utilizing these factors can aid in the development of cutting-edge mucosal vaccines that have the ability to elicit strong protective immune responses at the primary sites of pathogen invasion.

Surface Display of Duck Hepatitis A Virus Type 1 VP1 Protein on Bacillus subtilis Spores Elicits Specific Systemic and Mucosal Immune Responses on Mice.

Duck viral hepatitis, primarily caused by duck hepatitis A virus type 1 (DHAV-1), poses a significant threat to the global duck industry. Bacillus subtilis is commonly utilized as a safe probiotic in the development of mucosal vaccines. In this study, a recombinant strain of B. subtilis, designated as B. subtilis RV, was constructed to display the DHAV-1 capsid protein VP1 on its spore surface using the outer coat protein B as an anchoring agent. The immunogenicity of this recombinant strain was evaluated in a mouse model through mixed feeding immunization. The results indicated that B. subtilis RV could elicit specific systemic and mucosal immune responses in mice, as evidenced by the high levels of serum IgG, intestinal secretory IgA, and potent virus-neutralizing antibodies produced. Furthermore, the recombinant strain significantly upregulated the expression levels of IL-2, IL-6, IL-10, TNF-α, and IFN-γ in the intestinal mucosa. Thus, the recombinant strain maintained the balance of the Th1/Th2 immune response and demonstrated an excellent mucosal immune adjuvant function. In summary, this study suggests that B. subtilis RV can be a novel alternative for effectively controlling DHAV-1 infection as a vaccine-based feed additive.

Gut microbiota-derived metabolites tune host homeostasis fate.

The gut microbiota, housing trillions of microorganisms within the gastrointestinal tract, has emerged as a critical regulator of host health and homeostasis. Through complex metabolic interactions, these microorganisms produce a diverse range of metabolites that substantially impact various physiological processes within the host. This review aims to delve into the intricate relationships of gut microbiota-derived metabolites and their influence on the host homeostasis. We will explore how these metabolites affect crucial aspects of host physiology, including metabolism, mucosal integrity, and communication among gut tissues. Moreover, we will spotlight the potential therapeutic applications of targeting these metabolites to restore and sustain host equilibrium. Understanding the intricate interplay between gut microbiota and their metabolites is crucial for developing innovative strategies to promote wellbeing and improve outcomes of chronic diseases.

Effects of Hypoxia on the Antibacterial Activity of Epidermal Mucus from Chilean Meagre (Cilus gilberti).

Comprehending the immune defense mechanisms of new aquaculture species, such as the Chilean meagre (Cilus gilberti), is essential for sustaining large-scale production. Two bioassays were conducted to assess the impact of acute and intermittent hypoxia on the antibacterial activity of juvenile Chilean meagre epidermal mucus against the potential pathogens Vibrio anguillarum and Vibrio ordalii. Lysozyme and peroxidase activities were also measured. In general, fish exposed to hypoxia showed a 9-30% reduction in mucus antibacterial activity at the end of hypoxic periods and after stimulation with lipopolysaccharide. However, following water reoxygenation, the activity of non-stimulated fish was comparable to that of fish in normoxic conditions, inhibiting bacterial growth by 35-52%. In the case of fish exposed to chronic hypoxia, the response against V. anguillarum increased by an additional 19.8% after 6 days of control inoculation. Lysozyme exhibited a similar pattern, while no modulation of peroxidase activity was detected post-hypoxia. These results highlight the resilience of C. gilberti to dissolved oxygen fluctuations and contribute to understanding the potential of mucus in maintaining the health of cultured fish and the development of future control strategies.

Bromodomain-containing 4 is a positive regulator of the inflammatory cytokine response in the gut.

BACKGROUND AND AIM: Bromodomain-containing protein 4 (BRD4), one of the components of the bromodomain and extraterminal domain (BET) family, is a transcriptional and epigenetic regulator of cellular proliferation and cytokine production. In this study, we assessed whether BRD4 regulates the cytokine response in inflammatory bowel diseases (IBD). MATERIALS AND METHODS: BRD4 expression was analyzed in intestinal mucosal samples of patients with ulcerative colitis (UC), patients with Crohn's disease (CD), normal controls (CTRs), and mice with chemically-induced colitis by real-time PCR, Western blotting, and confocal microscopy. Cytokine production was evaluated in lamina propria mononuclear cells (LPMCs) of IBD patients and mucosal tissues of colitic mice treated with BRD4 inhibitors. Finally, we evaluated the effect of JQ1, an inhibitor of the BRD4 signaling pathway, on the course of murine colitis. RESULTS: BRD4 RNA and protein expression was up-regulated in the inflamed mucosa of patients with UC and patients with CD as compared to the uninvolved areas of the same patients and CTRs, and in the inflamed colon of colitic mice. Knockdown of BRD4 with a specific antisense oligonucleotide in IBD LPMCs led to reduced expression of TNF-α, IL-6, IFN-γ, and IL-17A. Administration of JQ1 to colitic mice inhibited the inflammatory cytokine response and attenuated the ongoing colitis. CONCLUSIONS: This is the first study showing the up-regulation of BRD4 in IBD and suggesting the role of such a protein in the positive control of the inflammatory cytokine response in the gut.

Weep and sweep and the broom: tuft cell acetylcholine limits the worm.

Two studies defined how tuft cell acetylcholine promotes parasite expulsion. Billip et al. demonstrated that acetylcholine increases water secretion, to promote the 'weep' response. Ndjim et al. found that tuft cell acetylcholine has a direct effect on worm fecundity. Both processes are only effective in the remodeled epithelium when the rare tuft cells have become abundant.

Modulation of metabolic and immunoregulatory pathways in the gut transcriptome of Atlantic salmon (Salmo salar L.) after early nutritional programming during first feeding with plant-based diet.

Introduction: Plant-based nutritional programming is the concept of exposing fish at very early life stages to a plant-based diet for a short duration to improve physiological responses when exposed to a similar plant-rich diet at a later developmental stage. The mechanisms of action underlying nutritional programming have not been fully deciphered, and the responses may be controlled at multiple levels. Methods: This 22-week study examines gut transcriptional changes after nutritional programming. Triplicate groups of Atlantic salmon were fed with a plant (V) vs. a marine-rich (M, control) diet for 2 weeks (stimulus phase) at the first exogenous feeding. Both stimulus fish groups (M and V fish) were then fed the M diet for 12 weeks (intermediate phase) and lastly fed the V diet (challenge phase) for 6 weeks, generating two dietary regimes (MMV and VMV) across phases. This study used a whole-transcriptome approach to analyse the effects of the V diet at the end of stimulus (short-term effects) and 22 weeks post-first feeding (long-term effects). After the stimulus, due to its developmental stage, the whole intestine was used, whereas, after the challenge, pyloric caeca and middle and distal intestines were examined. Results and discussion: At the stimulus end, genes with increased expression in V fish enriched pathways including regulatory epigenetic responses and lipid metabolism, and genes involved in innate immune response were downregulated. In the middle intestine at the end of the challenge, expression levels of genes of lipid, carbohydrate, and energy metabolism were increased in V fish, while M fish revealed increased expression of genes associated with autoimmune and acute adaptive immune response. The distal intestine of V fish showed increased expression of genes associated with immune response and potential immune tolerance. Conversely, the distal intestine of M fish at challenge revealed upregulation of lipid and carbohydrate metabolic pathways, tissue degeneration, and apoptotic responses. The present study demonstrated nutritional programming-associated changes in the intestinal transcriptome, with altered expression of genes involved in both immune responses and different metabolic processes. While there were limited changes in growth between the groups, the results show that there were transcriptional differences, suggesting a programming response, although the mechanism of this response still requires to be fully elucidated.

Protection from environmental enteric dysfunction and growth improvement in malnourished newborns by amplification of secretory IgA.

Environmental enteric dysfunction (EED) is a condition associated with malnutrition that can progress to malabsorption and villous atrophy. Severe EED results in linear growth stunting, slowed neurocognitive development, and unresponsiveness to oral vaccines. Prenatal exposure to malnutrition and breast feeding by malnourished mothers replicates EED. Pups are characterized by deprivation of secretory IgA (SIgA) and altered development of the gut immune system and microbiota. Extracellular ATP (eATP) released by microbiota limits T follicular helper (Tfh) cell activity and SIgA generation in Peyer's patches (PPs). Administration of a live biotherapeutic releasing the ATP-degrading enzyme apyrase to malnourished pups restores SIgA levels and ameliorates stunted growth. SIgA is instrumental in improving the growth and intestinal immune competence of mice while they are continuously fed a malnourished diet. The analysis of microbiota composition suggests that amplification of endogenous SIgA may exert a dominant function in correcting malnourishment dysbiosis and its consequences on host organisms, irrespective of the actual microbial ecology.

The role of immunoglobulin transport receptor, neonatal Fc receptor in mucosal infection and immunity and therapeutic intervention.

The neonatal Fc receptor (FcRn) can transport IgG and antigen-antibody complexes participating in mucosal immune responses that protect the host from most pathogens' invasion via the respiratory, digestive, and urogenital tracts. FcRn expression can be triggered upon stimulation with pathogenic invasion on mucosal surfaces, which may significantly modulate the innate immune response of the host. As an immunoglobulin transport receptor, FcRn is implicated in the pathophysiology of immune-related diseases such as infection and autoimmune disorders. In this review, we thoroughly summarize the recent advancement of FcRn in mucosal immunity and its therapeutic strategy. This includes insights into its regulation mechanisms of FcRn expression influenced by pathogens, its emerging role in mucosal immunity and its potential probability as a therapeutic target in infection and autoimmune diseases.

Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Virus-Mimetic Extracellular-Vesicle Vaccine Boosts Systemic and Mucosal Immunity via Immune Recruitment.

Mucosal vaccines can prevent viruses from infecting the respiratory mucosa, rather than only curtailing infection and protecting against the development of disease symptoms. The SARS-CoV-2 spike receptor-binding domain (RBD) is a compelling vaccine target but is undermined by suboptimal mucosal immunogenicity. Here, we report a SARS-CoV-2-mimetic extracellular-vesicle vaccine developed using genetic engineering and dendritic cell membrane budding. After mucosal immunization, the vaccine recruits antigen-presenting cells rapidly initiating a strong innate immune response. Notably, it obviates the need for adjuvants and can induce germinal center formation through both intramuscular and intratracheal vaccination. It not only elicits high levels of RBD-specific antibodies but also stimulates extensive cellular immunity in the respiratory mucosa. A sequential immunization strategy, starting with an intramuscular injection followed by an intratracheal booster, significantly bolsters mucosal immunity with high levels of IgA and tissue-resident memory T cell responses, thereby establishing a formidable defense against pseudovirus infection.

Eosinophils as drivers of bacterial immunomodulation and persistence.

Traditionally, eosinophils have been linked to parasitic infections and pathological disease states. However, emerging literature has unveiled a more nuanced and intricate role for these cells, demonstrating their key functions in maintaining mucosal homeostasis. Eosinophils exhibit diverse phenotypes and exert multifaceted effects during infections, ranging from promoting pathogen persistence to triggering allergic reactions. Our investigations primarily focus on Bordetella spp., with particular emphasis on Bordetella bronchiseptica, a natural murine pathogen that induces diseases in mice akin to pertussis in humans. Recent findings from our published work have unveiled a striking interaction between B. bronchiseptica and eosinophils, facilitated by the btrS-mediated mechanism. This interaction serves to enhance pathogen persistence while concurrently delaying adaptive immune responses. Notably, this role of eosinophils is only noted in the absence of a functional btrS signaling pathway, indicating that wild-type B. bronchiseptica, and possibly other Bordetella spp., possess such adeptness in manipulating eosinophils that the true function of these cells remains obscured during infection. In this review, we present the mounting evidence pointing toward eosinophils as targets of bacterial exploitation, facilitating pathogen persistence and fostering chronic infections in diverse mucosal sites, including the lungs, gut, and skin. We underscore the pivotal role of the master regulator of Bordetella pathogenesis, the sigma factor BtrS, in orchestrating eosinophil-dependent immunomodulation within the context of pulmonary infection. These putative convergent strategies of targeting eosinophils offer promising avenues for the development of novel therapeutics targeting respiratory and other mucosal pathogens.

Effects of High-Mobility Group Box-1 on Mucosal Immunity and Epithelial Differentiation in Colitic Carcinoma.

Abnormalities in mucosal immunity are involved in the onset and progression of ulcerative colitis (UC), resulting in a high incidence of colorectal cancer (CRC). While high-mobility group box-1 (HMGB1) is overexpressed during colorectal carcinogenesis, its role in UC-related carcinogenesis remains unclear. In the present study, we investigated the role of HMGB1 in UC-related carcinogenesis and sporadic CRC. Both the azoxymethane colon carcinogenesis and dextran sulfate sodium colitis carcinogenesis models demonstrated temporal increases in mucosal HMGB1 levels. Activated CD8+ cells initially increased and then decreased, whereas exhausted CD8+ cells increased. Additionally, we observed increased regulatory CD8+ cells, decreased naïve CD8+ cells, and decreased mucosal epithelial differentiation. In the in vitro study, HMGB1 induced energy reprogramming from oxidative phosphorylation to glycolysis in CD8+ cells and intestinal epithelial cells. Furthermore, in UC dysplasia, UC-related CRC, and hyperplastic mucosa surrounding human sporadic CRC, we found increased mucosal HMGB1, decreased activated CD8+ cells, and suppressed mucosal epithelial differentiation. However, we observed increased activated CD8+ cells in active UC mucosa. These findings indicate that HMGB1 plays an important role in modulating mucosal immunity and epithelial dedifferentiation in both UC-related carcinogenesis and sporadic CRC.

Development of a colloidal gold-based immunochromatographic assay for rapid detection of nasal mucosal secretory IgA against SARS-CoV-2.

Introduction: Infection with SARS-CoV-2 begins in the upper respiratory tract and can trigger the production of mucosal spike-specific secretory IgA (sIgA), which provides protection against reinfection. It has been recognized that individuals with high level of nasal spike-specific IgA have a lower risk of reinfection. However, mucosal spike-specific sIgA wanes over time, and different individuals may have various level of spike-specific sIgA and descending kinetics, leading to individual differences in susceptibility to reinfection. A method for detecting spike-specific sIgA in the nasal passage would be valuable for predicting the risk of reinfection so that people at risk can have better preparedness. Methods: In this study, we describe the development of a colloidal gold-based immunochromatographic (ICT) strip for detecting SARS-CoV-2 Omicron spike-specific sIgA in nasal mucosal lining fluids (NMLFs). Results: The ICT strip was designed to detect 0.125 µg or more spike-specific sIgA in 80 µL of NMLFs collected using a nasal swab. Purified nasal sIgA samples from individuals who recently recovered from an Omicron BA.5 infection were used to demonstrate that this ICT strip can specifically detect spike-specific sIgA. The signal levels positively correlated with neutralizing activities against XBB. Subsequent analysis revealed that people with low or undetectable levels of spike-specific sIgA in the nasal passage were more susceptible to SARS-CoV-2 reinfection. Conclusions: This nasal spike-specific sIgA ICT strip provides a non-invasive, rapid, and convenient method to assess the risk of reinfection for achieving precision preparedness.

The synergy of recombinant NSP4 and VP4 from porcine rotavirus elicited a strong mucosal response.

Porcine rotavirus (PoRV) is one of the main pathogens causing diarrhea in piglets, and multiple genotypes coexist. However, an effective vaccine is currently lacking. Here, the potential adjuvant of nonstructural protein 4 (NSP4) and highly immunogenic structural protein VP4 prompted us to construct recombinant NSP486-175aa (NSP4*) and VP426-476aa (VP4*) proteins, combine them as immunogens to evaluate their efficacy. Results indicated that NSP4* enhanced systemic and local mucosal responses induced by VP4*. The VP4*-IgG, VP4*-IgA in feces and IgA-secreting cells in intestines induced by the co-immunization were significantly higher than those induced by VP4* alone. Co-immunization of NSP4* and VP4* also induced strong cellular immunity with significantly increased IFN-λ than the single VP4*. Summarily, the NSP4* as a synergistical antigen exerted limited effects on the PoRV NAbs elevation, but conferred strong VP4*-specific mucosal and cellular efficacy, which lays the foundation for the development of a more effective porcine rotavirus subunit vaccine.

Deciphering the M-cell niche: insights from mouse models on how microfold cells "know" where they are needed.

Known for their distinct antigen-sampling abilities, microfold cells, or M cells, have been well characterized in the gut and other mucosa including the lungs and nasal-associated lymphoid tissue (NALT). More recently, however, they have been identified in tissues where they were not initially suspected to reside, which raises the following question: what external and internal factors dictate differentiation toward this specific role? In this discussion, we will focus on murine studies to determine how these cells are identified (e.g., markers and function) and ask the broader question of factors triggering M-cell localization and patterning. Then, through the consideration of unconventional M cells, which include villous M cells, Type II taste cells, and medullary thymic epithelial M cells (microfold mTECs), we will establish the M cell as not just a player in mucosal immunity but as a versatile niche cell that adapts to its home tissue. To this end, we will consider the lymphoid structure relationship and apical stimuli to better discuss how the differing cellular programming and the physical environment within each tissue yield these cells and their unique organization. Thus, by exploring this constellation of M cells, we hope to better understand the multifaceted nature of this cell in its different anatomical locales.

Fabrication of chitosan-based emulsion as an adjuvant to enhance nasal mucosal immune responses.

Nasal vaccine is a non-invasive vaccine that activates systemic and mucosal immunity in the presence of an adjuvant, thereby enhancing immune function. In this work, chitosan/oligochitosan/tween 80 (CS-COS-T80) co-stabilized emulsion was designed and further used as the nasal adjuvant. CS-COS-T80 emulsion exhibited outstanding stability under pH 6-8 with uniformly dispersed droplets and nano-scale particle size (<0.25 µm), and maintained stable at 4 °C for 150-day storage. Addition of model antigen ovalbumin (OVA) had no effect on the stability of CS-COS-T80 emulsion. In vivo nasal immunity indicated that CS-COS-T80 emulsion prolonged the retention time of OVA in the nasal cavity (from 4 to 8 h to >12 h), as compared to T80-emulsion. CS-COS-T80 emulsion produced a stronger mucosal immune response to OVA, with secretory IgA levels 5-fold and 2-fold higher than those of bare OVA and commercial adjuvant MF59, respectively. Compared to MF59, CS-COS-T80 induced a stronger humoral immune response and a mixed Th1/Th2 immune response of OVA after immunization. Furthermore, in the presence of CS-COS-T80 emulsion, the secretion of IL-4 and IFN-γ and the activation of splenocyte memory T-cell differentiation increased from 173.98 to 210.21 pg/mL and from 75.46 to 104.01 pg/mL, respectively. Therefore, CS-COS-T80 emulsion may serve as a promising adjuvant platform.

Immunogenicity of intranasal vaccine based on SARS-CoV-2 spike protein during primary and booster immunizations in mice.

Mucosal immunity plays a crucial role in combating and controlling the spread of highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recombinant subunit vaccines have shown safety and efficacy in clinical trials, but further investigation is necessary to evaluate their feasibility as mucosal vaccines. This study developed a SARS-CoV-2 mucosal vaccine using spike (S) proteins from a prototype strain and the omicron variant, along with a cationic chitosan adjuvant, and systematically evaluated its immunogenicity after both primary and booster immunization in mice. Primary immunization through intraperitoneal and intranasal administration of the S protein elicited cross-reactive antibodies against prototype strains, as well as delta and omicron variants, with particularly strong effects observed after mucosal vaccination. In the context of booster immunization following primary immunization with inactivated vaccines, the omicron-based S protein mucosal vaccine resulted in a broader and more robust neutralizing antibody response in both serum and respiratory mucosa compared to the prototype vaccine, enhancing protection against different variants. These findings indicate that mucosal vaccination with the S protein has the potential to trigger a broader and stronger antibody response during primary and booster immunization, making it a promising strategy against respiratory pathogens.

Detection of SARS-CoV-2-Specific Secretory IgA and Neutralizing Antibodies in the Nasal Secretions of Exposed Seronegative Individuals.

Mucosal immunity may contribute to clearing SARS-CoV-2 infection prior to systemic infection, thereby allowing hosts to remain seronegative. We describe the meaningful detection of SARS-CoV-2-specific nasal mucosal antibodies in a group of exposed-household individuals that evaded systemic infection. Between June 2020 and February 2023, nasopharyngeal swab (NPS) and acute and convalescent blood were collected from individuals exposed to a SARS-CoV-2-confirmed household member. Nasal secretory IgA (SIgA) antibodies targeting the SARS-CoV-2 spike protein were measured using a modified ELISA. Of the 36 exposed individuals without SARS-CoV-2 detected by the RT-PCR of NPS specimens and seronegative for SARS-CoV-2-specific IgG at enrollment and convalescence, 13 (36.1%) had positive SARS-CoV-2-specific SIgA levels detected in the nasal mucosa at enrollment. These individuals had significantly higher nasal SIgA (median 0.52 AU/mL) compared with never-exposed, never-infected controls (0.001 AU/mL) and infected-family participants (0.0002 AU/mL) during the acute visit, respectively (both p < 0.001). The nasal SARS-CoV-2-specific SIgA decreased rapidly over two weeks in the exposed seronegative individuals compared to a rise in SIgA in infected-family members. The nasal SARS-CoV-2-specific SIgA may have a protective role in preventing systemic infection.

mRNA Technology and Mucosal Immunization.

Current mRNA vaccines are mainly administered via intramuscular injection, which induces good systemic immunity but limited mucosal immunity. Achieving mucosal immunity through mRNA vaccination could diminish pathogen replication at the entry site and reduce interhuman transmission. However, delivering mRNA vaccines to mucosae faces challenges like mRNA degradation, poor entry into cells, and reactogenicity. Encapsulating mRNA in extracellular vesicles may protect the mRNA and reduce reactogenicity, making mucosal mRNA vaccines possible. Plant-derived extracellular vesicles from edible fruits have been investigated as mRNA carriers. Studies in animals show that mRNA vehiculated in orange-derived extracellular vesicles can elicit both systemic and mucosal immune responses when administered by the oral, nasal, or intramuscular routes. Once lyophilized, these products show remarkable stability. The optimization of mRNA to improve translation efficiency, immunogenicity, reactogenicity, and stability can be obtained through adjustments of the 5'cap region, poly-A tail, codons selection, and the use of nucleoside analogues. Recent studies have also proposed self-amplifying RNA vaccines containing an RNA polymerase as well as circular mRNA constructs. Data from parenterally primed animals demonstrate the efficacy of nasal immunization with non-adjuvanted protein, and studies in humans indicate that the combination of a parenteral vaccine with the natural exposure of mucosae to the same antigen provides protection and reduces transmission. Hence, mucosal mRNA vaccination would be beneficial at least in organisms pre-treated with parenteral vaccines. This practice could have wide applications for the treatment of infectious diseases.