Gut microbiota as a key regulator of intestinal mucosal immunity.

Gut microbiota is a complex microbial community with the ability of maintaining intestinal health. Intestinal homeostasis largely depends on the mucosal immune system to defense external pathogens and promote tissue repair. In recent years, growing evidence revealed the importance of gut microbiota in shaping intestinal mucosal immunity. Therefore, according to the existing findings, this review first provided an overview of intestinal mucosal immune system before summarizing the regulatory roles of gut microbiota in intestinal innate and adaptive immunity. Specifically, this review delved into the gut microbial interactions with the cells such as intestinal epithelial cells (IECs), macrophages, dendritic cells (DCs), neutrophils, and innate lymphoid cells (ILCs) in innate immunity, and T and B lymphocytes in adaptive immunity. Furthermore, this review discussed the main effects of gut microbiota dysbiosis in intestinal diseases and offered future research prospects. The review highlighted the key regulatory roles of gut microbiota in intestinal mucosal immunity via various host-microbe interactions, providing valuable references for the development of microbial therapy in intestinal diseases.

SARS-CoV-2-specific mucosal immune response in vaccinated versus infected children.

The anti-COVID-19 intramuscular vaccination induces a strong systemic but a weak mucosal immune response in adults. Little is known about the mucosal immune response in children infected or vaccinated against SARS-CoV-2. We found that 28% of children had detectable salivary IgA against SARS-CoV-2 even before vaccination, suggesting that, in children, SARS-CoV-2 infection may be undiagnosed. After vaccination, only receptor-binding domain (RBD)-specific IgA1 significantly increased in the saliva. Conversely, infected children had significantly higher salivary RBD-IgA2 compared to IgA1, indicating that infection more than vaccination induces a specific mucosal immune response in children. Future efforts should focus on development of vaccine technologies that also activate mucosal immunity.

The Nucleocapsid Protein of SARS-CoV-2, Combined with ODN-39M, Is a Potential Component for an Intranasal Bivalent Vaccine with Broader Functionality.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

SARS-CoV-2-Specific Immune Responses in Vaccination and Infection during the Pandemic in 2020-2022.

To gain insight into how immunity develops against SARS-CoV-2 from 2020 to 2022, we analyzed the immune response of a small group of university staff and students who were either infected or vaccinated. We investigated the levels of receptor-binding domain (RBD)-specific and nucleocapsid (N)-specific IgG and IgA antibodies in serum and saliva samples taken early (around 10 days after infection or vaccination) and later (around 1 month later), as well as N-specific T-cell responses. One patient who had been infected in 2020 developed serum RBD and N-specific IgG antibodies, but declined eight months later, then mRNA vaccination in 2021 produced a higher level of anti-RBD IgG than natural infection. In the vaccination of naïve individuals, vaccines induced anti-RBD IgG, but it declined after six months. A third vaccination boosted the IgG level again, albeit to a lower level than after the second. In 2022, when the Omicron variant became dominant, familial transmission occurred among vaccinated people. In infected individuals, the levels of serum anti-RBD IgG antibodies increased later, while anti-N IgG peaked earlier. The N-specific activated T cells expressing IFN γ or CD107a were detected only early. Although SARS-CoV-2-specific salivary IgA was undetectable, two individuals showed a temporary peak in RBD- and N-specific IgA antibodies in their saliva on the second day after infection. Our study, despite having a small sample size, revealed that SARS-CoV-2 infection triggers the expected immune responses against acute viral infections. Moreover, our findings suggest that the temporary mucosal immune responses induced early during infection may provide better protection than the currently available intramuscular vaccines.

SARS-CoV-2 Omicron infection augments the magnitude and durability of systemic and mucosal immunity in triple-dose CoronaVac recipients.

The inactivated whole-virion vaccine, CoronaVac, is one of the most widely used coronavirus disease 2019 (COVID-19) vaccines worldwide. There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron infection. In this prospective cohort study, 41 recipients of triple-dose CoronaVac and 14 unvaccinated individuals were recruited. We comprehensively profiled adaptive immune parameters in both groups, including spike-specific immunoglobulin (Ig) G and IgA titers, neutralizing activity, B cells, circulating follicular helper T (cTfh) cells, CD4+ and CD8+ T cells, and their memory subpopulations at 12 months after the third booster dose and at 4 and 20 weeks after Omicron BA.5 infection. Twelve months after the third CoronaVac vaccination, spike-specific antibodies and cellular responses were detectable in most vaccinated individuals. BA.5 infection significantly augmented the magnitude, cross-reactivity, and durability of serum neutralization activities, Fc-mediated phagocytosis, nasal spike-specific IgA responses, memory B cells, activated cTfh cells, memory CD4+ T cells, and memory CD8+ T cells for both the ancestral strain and Omicron subvariants, compared to unvaccinated individuals. Notably, the increase in BA.5-specific immunity after breakthrough infection was consistently comparable to or higher than that of the ancestral strain, suggesting no evidence of immune imprinting. Immune landscape analyses showed that vaccinated individuals have better synchronization of multiple immune components than unvaccinated individuals upon heterologous infection. Our data provide detailed insight into the protective role of the inactivated COVID-19 vaccine in shaping humoral and cellular immunity to Omicron infection. IMPORTANCE: There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron breakthrough infection. In this prospective cohort study, the anti-severe acute respiratory syndrome coronavirus 2 adaptive responses were analyzed before and after the Omicron BA.5 infection. Our data provide detailed insight into the protective role of the inactivated COVID-19 vaccine in shaping humoral and cellular immune responses to heterologous Omicron infection. CLINICAL TRIAL: This study is registered with ClinicalTrials.gov as NCT05680896.

Progress and challenges in the clinical evaluation of immune responses to respiratory mucosal vaccines.

INTRODUCTION: Following the coronavirus disease pandemic, respiratory mucosal vaccines that elicit both mucosal and systemic immune responses have garnered increasing attention. However, human physiological characteristics pose significant challenges in the evaluation of mucosal immunity, which directly impedes the development and application of respiratory mucosal vaccines. AREAS COVERED: This study summarizes the characteristics of immune responses in the respiratory mucosa and reviews the current status and challenges in evaluating immune response to respiratory mucosal vaccines. EXPERT OPINION: Secretory Immunoglobulin A (S-IgA) is a major effector molecule at mucosal sites and a commonly used indicator for evaluating respiratory mucosal vaccines. However, the unique physiological structure of the respiratory tract pose significant challenges for the clinical collection and detection of S-IgA. Therefore, it is imperative to develop a sampling method with high collection efficiency and acceptance, a sensitive detection method, reference materials for mucosal antibodies, and to establish a threshold for S-IgA that correlates with clinical protection. Sample collection is even more challenging when evaluating mucosal cell immunity. Therefore, a mucosal cell sampling method with high operability and high tolerance should be established. Targets of the circulatory system capable of reflecting mucosal cellular immunity should also be explored.

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Saccharomyces cerevisiae cells that display norovirus P induce both systemic and mucosal neutralizing antibodies.

The human norovirus (HuNov) is the leading cause of acute gastroenteritis (AGE) worldwide. Mucosal secretory IgA (sIgA) in the gastrointestinal tract interrupts the interaction between host cells and HuNov, thus inhibiting viral infection. In this study, we constructed a recombinant Saccharomyces cerevisiae (S. cerevisiae) expressing the HuNov P protein (GII. 4) and evaluated its immunogenicity in mice after oral delivery. First, the recombinant S. cerevisiae (EBY100/pYD1-P) efficiently expressed P, as evidenced by western blotting and indirect fluorescent assay. Second, after oral administration, EBY100/pYD1-P, especially the high-dose group (5 × 109 clone formation units), elicited systemic and mucosal immune responses characterized by significant sera IgG, IgA, and mucosal sIgA. More importantly, these antibodies showed a substantial neutralization effect against P. Lastly, EBY100/pYD1-P induced significant P-specific IFN-γ-secreting T cells and IL4-secreting T cells. Collectively, the recombinant S. cerevisiae expressing HuNov P is a promising mucosal vaccine candidate against HuNov.

Proof of concept in utilizing the peptidoglycan skeleton of pathogenic bacteria as antigen delivery platform for enhanced immune response.

Subunit vaccines are becoming increasingly important because of their safety and effectiveness. However, subunit vaccines often exhibit limited immunogenicity, necessitating the use of suitable adjuvants to elicit robust immune responses. In this study, we demonstrated for the first time that pathogenic bacteria can be prepared into a purified peptidoglycan skeleton without nucleic acids and proteins, presenting bacterium-like particles (pBLP). Our results showed that the peptidoglycan skeletons screened from four pathogens could activate Toll-like receptor1/2 receptors better than bacterium-like particles from Lactococcus lactis in macrophages. We observed that pBLP was safe in mouse models of multiple ages. Furthermore, pBLP improved the performance of two commercial vaccines in vivo. We confirmed that pBLP successfully loaded antigens onto the surface and proved to be an effective antigen delivery platform with enhanced antibody titers, antibody avidity, balanced subclass distribution, and mucosal immunity. These results indicate that the peptidoglycan skeleton of pathogenic bacteria represents a new strategy for developing subunit vaccine delivery systems.

Gut-associated lymphoid tissue: a microbiota-driven hub of B cell immunity.

The diverse gut microbiota, which is associated with mucosal health and general wellbeing, maintains gut-associated lymphoid tissues (GALT) in a chronically activated state, including sustainment of germinal centers in a context of high antigenic load. This influences the rules for B cell engagement with antigen and the potential consequences. Recent data have highlighted differences between GALT and other lymphoid tissues. For example, GALT propagates IgA responses against glycans that show signs of having been generated in germinal centers. Other findings suggest that humans are among those species where GALT supports the diversification, propagation, and possibly selection of systemic B cells. Here, we review novel findings that identify GALT as distinctive, and able to support these processes.

Role of trained innate immunity against mucosal cancer.

Mucosal tissues are frequent targets of both primary and metastatic cancers. This has highlighted the significance of both innate and adaptive anti-cancer immunity at mucosal sites. Trained innate immunity (TII) is an emerging concept defined as enhanced reactivity of innate leukocytes long after a previous stimulation that induces prolonged epigenetic, transcriptional, and metabolic changes. Trained innate leukocytes can respond to heterologous targets due to their lacking of antigen-specificity in most cases. Emerging experimental and clinical data suggest that certain microbes or their products induce TII in mucosal-associated innate leukocytes which endows heterologous anti-tumor innate immunity, in both prophylactic and therapeutic scenarios. In this mini-review, we summarize updated findings on the significance of TII in mucosal cancers. We also attempt to raise a few key questions critical to our further understanding on the roles of TII in mucosal cancers, and to the potential application of TII as anti-cancer strategy.

Nasal Immunization Using Chitosan Nanoparticles with Glycoprotein B of Murine Cytomegalovirus.

The use of nanoparticles as a delivery system for a specific antigen could solve many limitations of mucosal vaccine applications, such as low immunogenicity, or antigen protection and stabilization. In this study, we tested the ability of nasally administered chitosan nanoparticles loaded with glycoprotein B of murine cytomegalovirus to induce an immune response in an animal model. The choice of chitosan nanoparticle type was made by in vitro evaluation of sorption efficiency and antigen release. Three types of chitosan nanoparticles were prepared: crosslinked with tripolyphosphate, coated with hyaluronic acid, and in complex with polycaprolactone. The hydrodynamic size of the nanoparticles by dynamic light scattering, zeta potential, Fourier transform infrared spectroscopy, scanning electron microscopy, stability, loading efficiency, and release kinetics with ovalbumin were evaluated. Balb/c mice were immunized intranasally using the three-dose protocol with nanoparticles, gB, and adjuvants Poly(I:C) and CpG ODN. Subsequently, the humoral and cell-mediated antigen-specific immune response was determined. On the basis of the properties of the tested nanoparticles, the cross-linked nanoparticles were considered optimal for further investigation. The results show that nanoparticles with Poly(I:C) and with gB alone raised IgG antibody levels above the negative control. In the case of mucosal IgA, only gB alone weakly induced the production of IgA antibodies compared to saline-immunized mice. The number of activated cells increased slightly in mice immunized with nanoparticles and gB compared to those immunized with gB alone or to negative control. The results demonstrated that chitosan nanoparticles could have potential in the development of mucosal vaccines.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Cistanche deserticola polysaccharide-functionalized dendritic fibrous nano-silica as oral vaccine adjuvant delivery enhancing both the mucosal and systemic immunity.

Oral vaccines are a safe and convenient alternative to injected vaccines and have great potential to prevent major infectious diseases. However, the harsh gastrointestinal (GI) environment, mucus barriers, low immunogenicity, and lack of effective and safe mucosal adjuvants are the major challenges for oral vaccine delivery. In recent years, nanoparticle-based strategies have become attractive for improving oral vaccine delivery. Here, the dendritic fibrous nano-silica (DFNS) grafted with Cistanche deserticola polysaccharide (CDP) nanoparticles (CDP-DFNS) were prepared and investigated how to impact the immune responses. CDP-DFNS facilitated the antigen uptake in mouse bone marrow-derived dendritic cells (BMDCs), and induce the activation of DCs in vitro. Furthermore, in vivo experiments, the result showed that the uptake efficiency by Peyer's patches (PPs) of CDP-DFNS/BSA was the best. And CDP-DFNS/BSA then significantly activated the DCs in lamina propria (LP), and T/B cells in PPs and mesenteric lymph nodes (MLNs). Moreover, the memory T cell responses in later period of vaccination was stronger than other groups. In addition, CDP-DFNS/BSA enhanced BSA-specific antibody IgG, IgA production, and SIgA secretion, was effective at inducing a strong mixed Th1/Th2 response and mucosal antibody responses. These results indicated that CDP-DFNS deserves further consideration as an oral vaccine adjuvant delivery system.

Mucosal immune responses and protective efficacy elicited by oral administration AMP-ZnONPs-adjuvanted inactivated H9N2 virus in chickens.

The avian influenza virus is infected through the mucosal route, thus mucosal barrier defense is very important. While the inactivated H9N2 vaccine cannot achieve sufficient mucosal immunity, adjuvants are needed to induce mucosal and systemic immunity to prevent poultry from H9N2 influenza virus infection. Our previous study found that polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles (AMP-ZnONPs) had immune-enhancing effects in vitro. This study aimed to evaluate the mucosal immune responses of oral whole-inactivated H9N2 virus (WIV)+AMP-ZnONPs and its impact on the animal challenge protection, and the corresponding changes of pulmonary metabolomics after the second immunization. The results showed that compared to the WIV, the combined treatment of WIV and AMP-ZnONPs significantly enhanced the HI titer, IgG and specific sIgA levels, the number of goblet cells and intestinal epithelial lymphocytes (iIELs) as well as the expression of J-chain, polymeric immunoglobulin receptor (pIgR), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß). In viral attack experiments, WIV combing with AMP-ZnONPs effectively reduced lung damage and viral titers in throat swabs. Interestingly, significant changes of both the IgA intestinal immune network and PPAR pathway could also be found in the WIV+AMP-ZnONPs group compared to the non-infected group. Taken together, these findings suggest that AMP-ZnONPs can serve as a potential mucosal vaccine adjuvant, thereby avoiding adverse stress and corresponding costs caused by vaccine injection.

The protective effect of intranasal immunization with influenza virus recombinant adenovirus vaccine on mucosal and systemic immune response.

Influenza virus is a kind of virus that poses several hazards of animal and human health. Therefore, it is important to develop an effective vaccine to prevent influenza. To this end we successfully packaged recombinant adenovirus rAd-NP-M2e-GFP expressing multiple copies of influenza virus conserved antigens NP and M2e and packaged empty vector adenovirus rAd-GFP. The effect of rAd-NP-M2e-GFP on the activation of dendritic cell (DC) in vitro and in vivo was detected by intranasal immunization. The results showed that rAd-NP-M2e-GFP promoted the activation of DC in vitro and in vivo. After the primary immunization and booster immunization of mice through the nasal immune way, the results showed that rAd-NP-M2e-GFP induced enhanced local mucosal-specific T cell responses, increased the content of SIgA in broncho alveolar lavage fluids (BALF) and triggered the differentiation of B cells in the germinal center. It is proved that rAd-NP-M2e-GFP can significantly elicit mucosal immunity and systemic immune response. In addition, rAd-NP-M2e-GFP could effectively protect mice after H1N1 influenza virus challenge. To lay the foundation and provide reference for further development of influenza virus mucosal vaccine in the future.

Recombinant L. lactis vaccine LL-plSAM-WAE targeting four virulence factors provides mucosal immunity against H. pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastric disease. An efficient oral vaccine would be mucosa-targeted and offer defense against colonization of invasive infection in the digestive system. Proteolytic enzymes and acidic environment in the gastrointestinal tract (GT) can, however, reduce the effectiveness of oral vaccinations. For the creation of an edible vaccine, L. lactis has been proposed as a means of delivering vaccine antigens. RESULTS: We developed a plSAM (pNZ8148-SAM) that expresses a multiepitope vaccine antigen SAM-WAE containing Urease, HpaA, HSP60, and NAP extracellularly (named LL-plSAM-WAE) to increase the efficacy of oral vaccinations. We then investigated the immunogenicity of LL-plSAM-WAE in Balb/c mice. Mice that received LL-plSAM-WAE or SAM-WAE with adjuvant showed increased levels of antibodies against H. pylori, including IgG and sIgA, and resulted in significant reductions in H. pylori colonization. Furthermore, we show that SAM-WAE and LL-plSAM-WAE improved the capacity to target the vaccine to M cells. CONCLUSIONS: These findings suggest that recombinant L. lactis could be a promising oral mucosa vaccination for preventing H. pylori infection.

SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Fulvic acid modulates mucosal immunity in fish skin: Sustainable aquaculture solution or environmental risk factor?

This is the first study determining the effects of bath exposure to fulvic acid, a humic substance, on the skin mucosal immunity of rainbow trout (Oncorhynchus mykiss). Humic substances have recently been gaining attention for their increasing concentrations in aquatic ecosystems and their use as supplements in sustainable aquaculture. This study demonstrated that water exposure to fulvic acid at concentrations of 5 mg C/L and 50 mg C/L increased lysozyme and alkaline phosphatase activities in the mucus by approximately 2-fold and 2.5 to 3.2-fold, respectively. Furthermore, exposure to 50 mg C/L resulted in a 77.0% increase in mucosal immunoglobulin concentrations compared to the other groups. Importantly, all mucus samples demonstrated significant antibacterial activity against Yersinia ruckeri, with control mucus reducing bacterial growth by 44.5% and exposure to fulvic acid increasing this effect to 26.3%. Although these modulations show promise for application in aquaculture, alterations of the beneficial microbiota from long-term exposure in natural waters can be expected. Monitoring the rising concentrations of humic substances in natural water bodies is therefore urgently needed. Overall, this study represents the first investigation revealing the ability of humic substances to modulate skin mucosal immunity and the capacity to combat microorganisms.