The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies.

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3's Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

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

Background: Helicobacter 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. Methods: We 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. Results: In 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. Conclusion: H. 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.

IdeS, a secreted proteinase of Streptococcus pyogenes, is bound to a nuclease at the bacterial surface where it inactivates opsonizing IgG antibodies.

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab')2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis.

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.

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.

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.

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 IL1ß 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.

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.

Antibodies play a key role in the immune system. These proteins stick to harmful substances, such as bacteria and other disease-causing pathogens, marking them for destruction or blocking their attack. Antibodies are highly selective, and this ability has been used to target particular molecules in research, diagnostics and therapies. Typically, antibodies need to stick to a particular segment, or 'epitope', on the surface of a cell in order to trigger an immune response. Knowing where these regions are can help explain how these immune proteins work and aid the development of more effective drugs and diagnostic tools. One way to identify these sites is to measure the nano-distance between antibodies and other features on the cell surface. To do this, researchers take multiple images of the cell the antibody is attached to using light microscopy. Various statistical methods are then applied to create an 'average image' that has a higher resolution and can therefore be used to measure the distance between these two points more accurately. While this approach works on fixed shapes, like a perfect circle, it cannot handle human cells and bacteria which are less uniform and have more complex surfaces. Here, Kumra Ahnlide et al. have developed a new method called SiteLoc which can overcome this barrier. The method involves two fluorescent probes: one attached to a specific site on the cell's surface, and the other to the antibody or another molecule of interest. These two probes emit different colours when imaged with a fluorescent microscope. To cope with objects that have uneven surfaces, such as cells and bacteria, the two signals are transformed to 'follow' the same geometrical shape. The relative distance between them is then measured using statistical methods. Using this approach, Kumra Ahnlide et al. were able to identify epitopes on a bacterium, and measure distances on the surface of human red blood cells. The SiteLoc system could make it easier to develop antibody-based treatments and diagnostic tools. Furthermore, it could also be beneficial to the wider research community who could use it to probe other questions that require measuring nanoscale distances.

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.

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.