Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xvii, 278 pages) : color illustrations.

Dissertation/Thesis Chair

Nicholas J Mantis

Committee Members

Todd Gray, Pallavi Ghosh, Kathleen McDonough, William O'Connor

Keywords

Antibody, Immunity, Monoclonal, Peyer's patch, Salmonella enterica, Secretory Immunoglobulin A, Salmonella infections, Salmonella enteritidis, Immunization, Immunoglobulin A

Subject Categories

Immunology of Infectious Disease | Microbiology

Abstract

The Gram-negative bacterium, Salmonella enterica is a prominent etiologic agent of both diarrheal disease and enteric fever that encompasses over 2500 serovars, including S. Typhimurium (STm) and S. Typhi (STy). S. enterica is transmitted through contaminated food and water and, following ingestion, invades the gastrointestinal (GI) mucosa. The bacterium uses flagellar-based motility to target microfold (M) cells overlying gut-associated lymphoid tissues known as Peyer’s patches in the small intestine. Entry into Peyer’s patch tissues is a pivotal step in the infection process, as the bacterium can then disseminate systemically in the host. Given the rise in antibiotic resistance amongst S. enterica serovars and a lag in vaccine development, there is a push to investigate alternative biologics for therapeutic use. As such, the goal of my dissertation was to investigate the potential of passively administered antibodies to limit S. enterica infection, examining STm and STy as pathogens of interest. Secretory IgA (SIgA) is the primary antibody found in mucosal secretions that line the gastrointestinal tract. It is also the predominant antibody in breastmilk, where it affords maternal immunity to newborns. SIgA antibodies specific for S. enterica LPS are postulated to facilitate host protection by arresting bacterial motility, reducing type III secretion system (T3SS)-mediated entry into epithelial cells, and by preventing access to the epithelium through bacterial agglutination. One such IgA antibody is Sal4, a well-characterized monoclonal antibody (mAb) that binds the O-antigen region of STm lipopolysaccharide (LPS), specifically at the acetylated abequose moiety known as the O5 epitope. While a number of biological activities have been attributed to Sal4 in vitro, the possible mechanisms of protection by Sal4 in vivo have yet to be fully elucidated. In Chapter 3, I developed a mouse model to examine the potential of Sal4 to passively immunize animals against oral STm infection via reduced bacterial invasion into Peyer’s patches, the primary site of invasion for STm in the mouse gut. In this model, oral delivery of Sal4 IgA significantly blocked STm invasion into Peyer’s patch tissues of infected animals, which led us to explore the capacity of other antibody isotypes to impede STm pathogenesis. I evaluated an IgG1 variant of Sal4 and found that Sal4 IgG treatment arrested flagellar-based motility and blocked STm invasion into epithelial monolayers to the same extent as Sal4 IgA in vitro at equivalent concentrations. However, unlike Sal4 IgA, Sal4 IgG failed to prevent wildtype invasion into Peyer’s patches when employed in vivo. I determined that the diminished efficacy was at least in part due to instability in the mouse GI tract, as Sal4 IgG was readily degraded in adult simulated gastric fluid (SGF) in vitro and its activity was partially restored in vivo after buffering. As a follow up to our invasion studies, in Chapter 4 I sought to utilize our in vivo model to determine the window of protection of Sal4 by examining the kinetics of STm after administration, using human recombinant Sal4 SIgA. By isolating STm colony forming units (CFUs) from the fecal samples of infected animals over time, I demonstrated that Sal4 mAbs significantly reduce wildtype STm colonization 21 hours after infection. In our tracking studies, I determined that bacterial transit time in the mouse is rapid, with STm cells detected at the rectal end of the GI tract 60 minutes after gavage by immunohistochemistry (IHC). When provided at time of challenge, Sal4 mAbs localized STm cells to the mucus layer overlying the epithelium, compared to PBS control groups where STm cells were freely dispersed in the lumen. These data highlight a likely mechanism of elimination by SIgA. Based on these results, I conclude that Sal4 mAbs can facilitate clearance of colonized STm via entrapment, but that timing for protection is limited due to dilution in the lumen from accelerated GI movement. Predominantly, IgA found on mucosal surfaces is in the form of SIgA, a complex heavily glycosylated molecule made up of two monomers bound by joining (J) chain and associated with secretory component (SC). In Chapter 5, to further elucidate the importance of human SIgA in mucosal protection from STm, I tested humanized recombinant Sal4 SIgA, as well as dIgA and mIgA isoforms, in parallel in the Salmonella invasion model. I found that human Sal4 SIgA successfully prevented wildtype STm invasion into Peyer’s patches and promoted robust agglutination of STm in the small intestines of infected animals. Sal4 SIgA-induced aggregates were resistant to detection by IHC despite rigorous antigen retrieval methods. Based on these results, I hypothesized that Sal4 SIgA treatment induced the production of exopolysaccharide (EPS) in vivo, which was recapitulated in vitro as crystal violet (CV) staining of borosilicate glass tubes and determined to be cellulose-dependent. Taken together, these data validate the efficacy of oral Sal4 SIgA to passively immunize the mucosa from STm infection and postulate agglutination and extracellular matrix induction as protective mechanisms by secretory antibodies. In addition to facilitating typhoid fever, STy produces an A2B5 toxin intracellularly following internalization into host cells that contributes to STy pathogenesis and disease progression. In Chapter 6, to examine the potential of antibodies to passively immunize against typhoid toxin, I developed a panel of 11 IgG mAbs by immunizing mice intraperitoneally with recombinant typhoid toxoid and generating hybridoma cell lines from antigen-specific splenic B cells. We found that the majority (10) of the mAbs bound the binding subunit, PltB, and identified three distinct hybridoma clones; TyTx1, 3, and 4. Each TyTx mAb was then evaluated in vitro by our collaborators at Cornell University for the ability to block toxin binding to human epithelial cells. TyTx 1 and 4 bound separate epitopes of PltB, which accounted for differences in antibody avidity, cell surface binding inhibition, and efficacy in vivo. Overall, these data demonstrate that TyTx mAbs passively protect against intoxication by typhoid toxin through multiple mechanisms and provide important insight into the future design of antibody-based intervention strategies. The results of this dissertation highlight the ability of mAbs to passively immunize against multiple serovars of S. enterica, by either arresting bacterial access in the lumen, or through direct toxin neutralization. Indeed, while oral delivery of SIgA protected the GALT from STm infection, the window of protection was determined to be limited. It is evident from these studies that robust agglutination by SIgA antibodies is required for protection from STm, which empathizes the need for adequate local concentrations of antibody at the time of infection. In the advent of transitioning these biologics for use in humans, sufficient dosing and stabilization of SIgA mAbs would be a prerequisite. Taken together, these studies emphasize the capacity of mAbs to defend against an arsenal of virulence factors that enteric bacterial pathogens may employ and define the challenges that must be overcome for future therapeutic use in humans.

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