ZMapp reduces diffusion of Ebola viral particles in fresh human cervicovaginal mucus.

Vaginal transmission from semen of male Ebola virus (EBOV) survivors has been implicated as a potential origin of Ebola virus disease (EVD) outbreaks. While EBOV in semen must traverse cervicovaginal mucus (CVM) to reach target cells, the behaviour of EBOV in CVM is poorly understood. CVM contains substantial quantities of IgG, and arrays of IgG bound to a virion can develop multiple Fc-mucin bonds, immobilizing the IgG/virion complex in mucus. Here, we measured the real-time mobility of fluorescent Ebola virus-like-particles (VLP) in 50 CVM specimens from 17 women, with and without ZMapp, a cocktail of 3 monoclonal IgGs against EBOV. ZMapp-mediated effective trapping of Ebola VLPs in CVM from a subset of women across the menstrual cycle, primarily those with Lactobacillus crispatus dominant microbiota. Our work underscores the influence of the vaginal microbiome on IgG-mucin crosslinking against EBOV and identifies bottlenecks in the sexual transmission of EBOV.

Multidisciplinary evidence-based tools for improving consistency of care and neonatal nutrition.

BACKGROUND: Extrauterine growth restriction from inadequate nutrition remains a significant morbidity in very low birth weight infants. Participants in the California Perinatal Quality Care Collaborative Quality Improvement Collaborative, Grow, Babies, Grow! developed or refined tools to improve nutrition and reduce practice variation. METHOD: Five Neonatal Intensive Care Units describe the development and implementation of nutrition tools. Tools include Parenteral Nutrition Guidelines, Automated Feeding Protocol, electronic medical record Order Set, Nutrition Time-Out Rounding Tool, and a Discharge Nutrition Recommendations. 15 of 22 participant sites completed a survey regarding tool value and implementation. RESULTS: Reduced growth failure at discharge was observed in four of five NICUs, 11-32% improvement. Tools assisted with earlier TPN initiation (8 h) and reaching full feeds (2-5 days). TPN support decreased by 5 days. 80% of survey respondents rated the tools as valuable. CONCLUSION: Evidence and consensus-based nutrition tools help promote standardization, leading to improved and sustainable outcomes.

Broadly neutralizing antibodies consistently trap HIV-1 in fresh cervicovaginal mucus from select individuals.

In addition to direct neutralization and other classical effector functions, IgG possesses a little recognized and thus under-utilized effector function at mucosal surfaces: Fc-mucin bonds enable IgG to trap viruses in mucus. Due to the paucity of envelope glycoproteins that limits the number of IgG that can bind HIV, it remains poorly understood whether IgG-mucin interactions can effectively immobilize HIV in human cervicovaginal mucus (CVM). Here, we obtained 54 fresh, undiluted CVM specimens from 17 different women, and employed high-resolution multiple particle tracking to quantify the mobility of fluorescent HIV virus-like-particles in CVM treated with various HIV-specific IgG. We observed consistent and effective trapping of HIV by broadly neutralizing antibodies (VRC01, PGT121, and 2F5) in a subset of women. While trapping efficacy was not affected by the menstrual cycle, it was positively correlated with appreciable L. Crispatus populations in the microbiome, and negatively correlated with appreciable L. Iners or G. Vaginalis populations. Our work demonstrates for the first time that IgG-mucin crosslinking is capable of reinforcing the mucosal barrier against HIV, and motivates further investigation of passive immunization against vaginal transmission of STIs. STATEMENT OF SIGNIFICANCE: HIV transmission in women primarily occurs vaginally, yet the 3-way interactions between mucins and HIV virions mediated by HIV-binding antibodies in cervicovaginal mucus (CVM) is not well understood. While IgG-Fc possess weak affinity to mucins that trap virus/IgG complexes in mucus, the effectiveness against HIV remains unclear, due to the low number of virion-bound IgG. Here, we discovered that IgG can trap HIV consistently in CVM from select individuals regardless of their birth control status or menstrual cycle phase. IgG-mediated trapping of HIV was moderately associated with microbiome composition. These results suggest that IgG-mucin interactions could potentially reduce HIV transmission and highlight the importance of mucosal secretions in antibody-mediated prevention of HIV and other sexually transmitted infections.

LPS-binding IgG arrests actively motile Salmonella Typhimurium in gastrointestinal mucus.

The gastrointestinal (GI) mucosa is coated with a continuously secreted mucus layer that serves as the first line of defense against invading enteric bacteria. We have previously shown that antigen-specific immunoglobulin G (IgG) can immobilize viruses in both human airway and genital mucus secretions through multiple low-affinity bonds between the array of virion-bound IgG and mucins, thereby facilitating their rapid elimination from mucosal surfaces and preventing mucosal transmission. Nevertheless, it remains unclear whether weak IgG-mucin crosslinks could reinforce the mucus barrier against the permeation of bacteria driven by active flagella beating, or in predominantly MUC2 mucus gel. Here, we performed high-resolution multiple particle tracking to capture the real-time motion of hundreds of individual fluorescent Salmonella Typhimurium in fresh, undiluted GI mucus from Rag1-/- mice, and analyzed the motion using a hidden Markov model framework. In contrast to control IgG, the addition of anti-lipopolysaccharide IgG to GI mucus markedly reduced the progressive motility of Salmonella by lowering the swim speed and retaining individual bacteria in an undirected motion state. Effective crosslinking of Salmonella to mucins was dependent on Fc N-glycans. Our findings implicate IgG-mucin crosslinking as a broadly conserved function that reduces mucous penetration of both bacterial and viral pathogens.

Modeling Barrier Properties of Intestinal Mucus Reinforced with IgG and Secretory IgA against Motile Bacteria.

The gastrointestinal (GI) tract is lined with a layer of viscoelastic mucus gel, characterized by a dense network of entangled and cross-linked mucins together with an abundance of antibodies (Ab). Secretory IgA (sIgA), the predominant Ab isotype in the GI tract, is a dimeric molecule with 4 antigen-binding domains capable of inducing efficient clumping of bacteria, or agglutination. IgG, another common Ab at mucosal surfaces, can cross-link individual viruses to the mucin mesh through multiple weak bonds between IgG-Fc and mucins, a process termed muco-trapping. Relative contributions by agglutination versus muco-trapping in blocking permeation of motile bacteria through mucus remain poorly understood. Here, we developed a mathematical model that takes into account physiologically relevant spatial dimensions and time scales, binding and unbinding rates between Ab and bacteria as well as between Ab and mucins, the diffusivities of Ab, and run-tumble motion of active bacteria. Our model predicts both sIgA and IgG can accumulate on the surface of individual bacteria at sufficient quantities and rates to enable trapping individual bacteria in mucins before they penetrate the mucus layer. Furthermore, our model predicts that agglutination only modestly improves the ability for antibodies to block bacteria permeation through mucus. These results suggest that while sIgA is the most potent Ab isotype overall at stopping bacterial penetration, IgG may represent a practical alternative for mucosal prophylaxis and therapy. Our work improves the mechanistic understanding of Ab-enhanced barrier properties of mucus and highlights the ability for muco-trapping Ab to protect against motile pathogens at mucosal surfaces.

Herpes simplex virus-binding IgG traps HSV in human cervicovaginal mucus across the menstrual cycle and diverse vaginal microbial composition.

IgG possesses an important yet little recognized effector function in mucus. IgG bound to viral surface can immobilize otherwise readily diffusive viruses to the mucin matrix, excluding them from contacting target cells and facilitating their elimination by natural mucus clearance mechanisms. Cervicovaginal mucus (CVM) is populated by a microbial community, and its viscoelastic and barrier properties can vary substantially not only across the menstrual cycle, but also in women with distinct microbiota. How these variations impact the "muco-trapping" effector function of IgGs remains poorly understood. Here we obtained multiple fresh, undiluted CVM specimens (n = 82 unique specimens) from six women over time, and employed high-resolution multiple particle tracking to quantify the mobility of fluorescent Herpes Simplex Viruses (HSV-1) in CVM treated with different HSV-1-binding IgG. The IgG trapping potency was then correlated to the menstrual cycle, and the vaginal microbial composition was determined by 16 s rRNA. In the specimens studied, both polyclonal and monoclonal HSV-1-binding IgG appeared to consistently and effectively trap HSV-1 in CVM obtained at different times of the menstrual cycle and containing a diverse spectrum of commensals, including G. vaginalis-dominant microbiota. Our findings underscore the potential broad utility of this "muco-trapping" effector function of IgG to reinforce the vaginal mucosal defense, and motivates further investigation of passive immunization of the vagina as a strategy to protect against vaginally transmitted infections.

Diffusion of Immunoglobulin G in Shed Vaginal Epithelial Cells and in Cell-Free Regions of Human Cervicovaginal Mucus.

Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments.

Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus.

UNLABELLED: Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces. STATEMENT OF SIGNIFICANCE: PEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.