Exploring Monoclonal Antibody Action Against the Group A Streptococcal M Protein

Group A Streptococcus (GAS) is a significant human pathogen that has developed multipleimmune evasion mechanisms to counter the host immune response. One of these mechanisms involvesthe production of the M protein, which, amongst other things, acts as an anti-phagocytic factor and canbind host proteins. Another is the ability of M protein to bind a human protein called fibronectin (Fn). Thisprotein plays a key role in a number of physiological processes and can be used by GAS to evade theimmune system. In this PhD dissertation, we aimed to assess the binding efficacy and function ofmonoclonal antibodies targeting the GAS M protein.In the first paper we start by developing a robust method to assess phagocytosis. This method highlightsthe importance of factors such as volume, time, and the ratio of phagocyte to prey on the phagocyticprocess. It has allowed us to, henceforth, attain precise, high quality phagocytosis data and has been amajor driving force for other projects within the lab – especially the three other papers included in thisthesis.In the second paper we discovered a novel form of antibody binding whereby a monoclonal binds theGAS M protein in a bivalent dual-Fab cis mode. This means that both Fab arms of the Ab bind to distinctepitopes on the target molecule simultaneously. Even so this antibody bound to a region of the M proteinassociated with non-opsonic antibodies we found that this Ab could enhance phagocytosis suggestingthat this novel binding form can circumvent the M protein's anti-phagocytic properties.In the third paper we investigated the M protein’s ability to bind fibronectin. While this function wasdescribed in previous studies, we found it could only do so with very low affinity. We found that the bindingof antibodies from the blood of donors who had recently recovered from a severe GAS infection couldgreatly enhance this fibronectin binding. We show that same occurs with certain anti-M monoclonals andthat this mechanism leads to a reduction in opsonophagocytosis. Moreover we find that Ab flexibility mayplay a role and that Ab Fc domains are a crucial factor in mechanism.In the fourth paper we further explore this anti-phagocytic effect. Here we assess the effects of varyingconcentrations of Fn since this can differ greatly within the human body. We found that both very low andhigh concentrations of Fn, corresponding with the nasopharyngeal niche and blood respectively, led to asubstantial reduction in phagocytosis. We moreover found that this reduction in phagocytosis is likelylinked to a modulation of integrins. Overall, this work provides insights into immune evasion mechanismsdeveloped by GAS and highlights how this remarkable pathogen always seems to be one step ahead ofus

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

BackgroundHelicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection.MethodsWe analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines.ResultsIn biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells.ConclusionH. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy

High-Sensitivity Assessment of Phagocytosis by Persistent Association-Based Normalization

Phagocytosis is measured as a functional outcome in many research fields, but accurate quantification can be challenging, with no robust method available for cross-laboratory reproducibility. In this study, we identified a simple, measurable parameter, persistent prey-phagocyte association, to use for normalization and dose-response analysis. We apply this in a straightforward analytical method, persistent association-based normalization, in which the multiplicity of prey (MOP) ratio needed to elicit half of the phagocytes to associate persistently (MOP50) is determined first. MOP50 is then applied to normalize for experimental factors, separately analyzing association and internalization. We use reference human phagocyte THP-1 cells with different prey and opsonization conditions to compare the persistent association-based normalization method to standard ways of assessing phagocytosis and find it to perform better, exhibiting increased robustness, sensitivity, and reproducibility. The approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity

Group A streptococci induce stronger M protein-fibronectin interaction when specific human antibodies are bound

Group A streptococcus (GAS) is a highly adapted, human-specific pathogen that is known to manipulate the immune system through various mechanisms. GAS’ M protein constitutes a primary target of the immune system due to its spatial configuration and dominance on the bacterial surface. Antibody responses targeting the M protein have been shown to favor the conserved C region. Such antibodies (Abs) circumvent antigenic escape and efficiently bind to various M types. The ability of GAS to bind to fibronectin (Fn), a high molecular weight glycoprotein of the extracellular matrix, has long been known to be essential for the pathogen’s evolutionary success and fitness. However, some strains lack the ability to efficiently bind Fn. Instead, they have been found to additionally bind Fn via the A-B domains of their M proteins. Here, we show that human Abs can induce increased Fn-binding affinity in M proteins, likely by enhancing the weak A-B domain binding. We found that this enhanced Fn binding leads to a reduction in Ab-mediated phagocytosis, indicating that this constitutes a GAS immune escape mechanism. We could show that the Fc domain of Abs is necessary to trigger this phenomenon and that Ab flexibility may also play a key role. We, moreover, saw that our Abs could enhance Fn binding in 3 out of 5 emm type strains tested, belonging to different clades, making it likely that this is a more generalizable phenomenon. Together our results suggest a novel synergistic interplay of GAS and host proteins which ultimately benefits the bacterium

Nanoscale binding site localization by molecular distance estimation on native cell surfaces using topological image averaging

Antibody binding to cell surface proteins plays a crucial role in immunity, and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines. Cellular morphology typically varies, and antibodies often bind heterogeneously across a cell surface, making traditional particle-averaging strategies challenging for accurate native antibody localization. In the present work, we have developed a method, SiteLoc, for imaging-based molecular localization on cellular surface proteins. Nanometer-scale resolution is achieved through localization in one dimension, namely, the distance from a bound ligand to a reference surface. This is done by using topological image averaging. Our results show that this method is well suited for antibody binding site measurements on native cell surface morphology and that it can be applied to other molecular distance estimations as well

Short-term exposure to urban PM2.5 particles induces histopathological and inflammatory changes in the rat small intestine

Air pollution and exposure to fine airborne particles with aerodynamic diameter <2.5 μm (PM2.5) negatively impacts human health. Airways constitute a primary route of exposure but PM2.5-contaminated food, drinks as well as mucociliary and hepatobiliary clearance all constitute potential entry points into the intestine. This study evaluated intestinal histopathological and inflammatory changes as well as enteric neuronal numbers after short- or long-term exposure to urban PM2.5. Using a nebulizer, male rats were exposed to a mist with a concentration of 5.3mg PM2.5/m3 for 8 h (short term) or 1.8 mg PM2.5/m3 for 3 h/day, 5 days/week for 8 weeks (long-term) with controls run in parallel. Samples were taken from three regions of the small intestine as well as the colon. Results showed that short-term exposure to PM2.5 induces mucosal lesions and reduces IL1β levels in the small intestine but not colon. No significant changes were observed after long-term exposure, suggesting the presence of intestinal adaptation to environmental stressors in the PM2.5. To our knowledge, this is the first study to systematically characterize regional effects along the intestine

Subclass-switched anti-Spike IgG3 oligoclonal cocktails strongly enhance Fc-mediated opsonization

Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an in vivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses

A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function

Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein's virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, regions for protective antibody targeting

Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2

Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization correlate with the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phagocytosis