An mRNA-based platform for the delivery of pathogen-specific IgA into mucosal secretions.

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.

Transcytosis of IgA Attenuates Salmonella Invasion in Human Enteroids and Intestinal Organoids.

Secretory IgA (SIgA) is the most abundant antibody type in intestinal secretions where it contributes to safeguarding the epithelium from invasive pathogens like the Gram-negative bacterium, Salmonella enterica serovar Typhimurium (STm). For example, we recently reported that passive oral administration of the recombinant monoclonal SIgA antibody, Sal4, to mice promotes STm agglutination in the intestinal lumen and restricts bacterial invasion of Peyer's patch tissues. In this report, we sought to recapitulate Sal4-mediated protection against STm in human Enteroids and human intestinal organoids (HIOs) as models to decipher the molecular mechanisms by which antibodies function in mucosal immunity in the human gastrointestinal tract. We confirm that Enteroids and HIO-derived monolayers are permissive to STm infection, dependent on HilD, the master transcriptional regulator of the SPI-I type three secretion system (T3SS). Stimulation of M-like cells in both Enteroids and HIOs by the addition of RANKL further enhanced STm invasion. The apical addition of Sal4 mouse IgA, as well as recombinant human Sal4 dimeric IgA (dIgA) and SIgA resulted a dose-dependent reduction in bacterial invasion. Moreover, basolateral application of Sal4 dIgA to Enteroid and HIO monolayers gave rise to SIgA in the apical compartment via a pathway dependent on expression of the polymeric immunoglobulin receptor (pIgR). The resulting Sal4 SIgA was sufficient to reduce STm invasion of Enteroid and HIO epithelial cell monolayers by ~20-fold. Recombinant Sal4 IgG was also transported in the Enteroid and HIOs, but to a lesser degree and via a pathway dependent on the neonatal Fc receptor (FCGRT). The models described lay the foundation for future studies into detailed mechanisms of IgA and IgG protection against STm and other pathogens.

Salmonella Uptake into Gut-Associated Lymphoid Tissues: Implications for Targeted Mucosal Vaccine Design and Delivery.

Peyer's patches are organized gut-associated lymphoid tissues (GALT) in the small intestine and the primary route by which particulate antigens, including viruses and bacteria, are sampled by the mucosal immune system. Antigen sampling occurs through M cells, a specialized epithelial cell type located in the follicle-associated epithelium (FAE) that overlie Peyer's patch lymphoid follicles. While Peyer's patches play an integral role in intestinal homeostasis, they are also a gateway by which enteric pathogens, like Salmonella enterica serovar Typhimurium (STm), cross the intestinal barrier. Once pathogens like STm gain access to the underlying network of mucosal dendritic cells and macrophages they can spread systemically. Thus, Peyer's patches are at the crossroads of mucosal immunity and intestinal pathogenesis. In this chapter, we provide detailed methods to assess STm entry into mouse Peyer's patch tissues. We describe Peyer's patch collection methods and provide strategies to enumerate bacterial uptake. We also detail a method for quantifying bacterial shedding from infected animals and provide an immunohistochemistry protocol for the localization of STm along the gastrointestinal tract and insight into pathogen transit in the presence of protective antibodies. While the protocols are written for STm, they are easily tailored to other enteric pathogens.

The prospect of orally administered monoclonal secretory IgA (SIgA) antibodies to prevent enteric bacterial infections.

Eliminating diarrheal diseases as a leading cause of childhood morbidity and mortality in low- and middle-income countries (LMICs) will require multiple intervention strategies. In this review, we spotlight a series of preclinical studies investigating the potential of orally administered monoclonal secretory IgA (SIgA) antibodies (MAbs) to reduce disease associated with three enteric bacterial pathogens: Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), and invasive Salmonella enterica serovar Typhimurium. IgA MAbs targeting bacterial surface antigens (flagella, adhesins, and lipopolysaccharide) were generated from mice, humanized mice, and human tonsillar B cells. Recombinant SIgA1 and/or SIgA2 derivates of those MAbs were purified from supernatants following transient transfection of 293 cells with plasmids encoding antibody heavy and light chains, J-chain, and secretory component (SC). When administered to mice by gavage immediately prior to (or admixed with) the bacterial challenge, SIgA MAbs reduced infection C. jejuni, ETEC, and S. Typhimurium infections. Fv-matched IgG1 MAbs by comparison were largely ineffective against C. jejuni and S. Typhimurium under the same conditions, although they were partially effective against ETEC. While these findings highlight future applications of orally administered SIgA, the studies also underscored the fundamental challenges associated with using MAbs as prophylactic tools against enteric bacterial diseases.

The structural basis of Salmonella A2B5 toxin neutralization by antibodies targeting the glycan-receptor binding subunits.

Many bacterial pathogens secrete A(2)B5 toxins comprising two functionally distinct yet complementary "A" and "B" subunits to benefit the pathogens during infection. The lectin-like pentameric B subunits recognize specific sets of host glycans to deliver the toxin into target host cells. Here, we offer the molecular mechanism by which neutralizing antibodies, which have the potential to bind to all glycan-receptor binding sites and thus completely inhibit toxin binding to host cells, are inhibited from exerting this action. Cryogenic electron microscopy (cryo-EM)-based analyses indicate that the skewed positioning of the toxin A subunit(s) toward one side of the toxin B pentamer inhibited neutralizing antibody binding to the laterally located epitopes, rendering some glycan-receptor binding sites that remained available for the toxin binding and endocytosis process, which is strikingly different from the counterpart antibodies recognizing the far side-located epitopes. These results highlight additional features of the toxin-antibody interactions and offer important insights into anti-toxin strategies.

Mechanisms of typhoid toxin neutralization by antibodies targeting glycan receptor binding and nuclease subunits.

Nearly all clinical isolates of Salmonella Typhi, the cause of typhoid fever, are antibiotic resistant. All S. Typhi isolates secrete an A2B5 exotoxin called typhoid toxin to benefit the pathogen during infection. Here, we demonstrate that antibiotic-resistant S. Typhi secretes typhoid toxin continuously during infection regardless of antibiotic treatment. We characterize typhoid toxin antibodies targeting glycan-receptor-binding PltB or nuclease CdtB, which neutralize typhoid toxin in vitro and in vivo, as demonstrated by using typhoid toxin secreted by antibiotic-resistant S. Typhi during human cell infection and lethal dose typhoid toxin challenge to mice. TyTx11 generated in this study neutralizes typhoid toxin effectively, comparable to TyTx4 that binds to all PltB subunits available per holotoxin. Cryoelectron microscopy explains that the binding of TyTx11 to CdtB makes this subunit inactive through CdtB catalytic-site conformational change. The identified toxin-neutralizing epitopes are conserved across all S. Typhi clinical isolates, offering critical insights into typhoid toxin-neutralizing strategies.

Recombinant Human Secretory IgA Induces Salmonella Typhimurium Agglutination and Limits Bacterial Invasion into Gut-Associated Lymphoid Tissues.

As the predominant antibody type in mucosal secretions, human colostrum, and breast milk, secretory IgA (SIgA) plays a central role in safeguarding the intestinal epithelium of newborns from invasive enteric pathogens like the Gram-negative bacterium Salmonella enterica serovar Typhimurium (STm). SIgA is a complex molecule, consisting of an assemblage of two or more IgA monomers, joining (J)-chain, and secretory component (SC), whose exact functions in neutralizing pathogens are only beginning to be elucidated. In this study, we produced and characterized a recombinant human SIgA variant of Sal4, a well-characterized monoclonal antibody (mAb) specific for the O5-antigen of STm lipopolysaccharide (LPS). We demonstrate by flow cytometry, light microscopy, and fluorescence microscopy that Sal4 SIgA promotes the formation of large, densely packed bacterial aggregates in vitro. In a mouse model, passive oral administration of Sal4 SIgA was sufficient to entrap STm within the intestinal lumen and reduce bacterial invasion into gut-associated lymphoid tissues by several orders of magnitude. Bacterial aggregates induced by Sal4 SIgA treatment in the intestinal lumen were recalcitrant to immunohistochemical staining, suggesting the bacteria were encased in a protective capsule. Indeed, a crystal violet staining assay demonstrated that STm secretes an extracellular matrix enriched in cellulose following even short exposures to Sal4 SIgA. Collectively, these results demonstrate that recombinant human SIgA recapitulates key biological activities associated with mucosal immunity and raises the prospect of oral passive immunization to combat enteric diseases.

Inhibition of invasive salmonella by orally administered IgA and IgG monoclonal antibodies.

Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.

MYOSLID Is a Novel Serum Response Factor-Dependent Long Noncoding RNA That Amplifies the Vascular Smooth Muscle Differentiation Program.

OBJECTIVE: Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, an lncRNA that seems to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the VSMC-specific lncRNAs regulated by myocardin (MYOCD)/serum response factor, the master switch for VSMC differentiation, is unknown. APPROACH AND RESULTS: To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs that overexpress MYOCD. Several novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced Smooth muscle LncRNA, Inducer of Differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/serum response factor and transforming growth factor-ß/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula samples compared with healthy veins. Although MYOSLID did not affect gene expression of transcription factors, such as serum response factor and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated transforming growth factor-ß1-induced SMAD2 phosphorylation. CONCLUSIONS: We have demonstrated that MYOSLID, the first human VSMC-selective and serum response factor/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and transforming growth factor-ß/SMAD pathways.