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.

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.

Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies.

Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2-) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy.IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

ZMapp Reinforces the Airway Mucosal Barrier Against Ebola Virus.

Filoviruses, including Ebola, have the potential to be transmitted via virus-laden droplets deposited onto mucus membranes. Protecting against such emerging pathogens will require understanding how they may transmit at mucosal surfaces and developing strategies to reinforce the airway mucus barrier. Here, we prepared Ebola pseudovirus (with Zaire strain glycoproteins) and used high-resolution multiple-particle tracking to track the motions of hundreds of individual pseudoviruses in fresh and undiluted human airway mucus isolated from extubated endotracheal tubes. We found that Ebola pseudovirus readily penetrates human airway mucus. Addition of ZMapp, a cocktail of Ebola-binding immunoglobulin G antibodies, effectively reduced mobility of Ebola pseudovirus in the same mucus secretions. Topical delivery of ZMapp to the mouse airways also facilitated rapid elimination of Ebola pseudovirus. Our work demonstrates that antibodies can immobilize virions in airway mucus and reduce access to the airway epithelium, highlighting topical delivery of pathogen-specific antibodies to the lungs as a potential prophylactic or therapeutic approach against emerging viruses or biowarfare agents.

Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota.

UNLABELLED: Cervicovaginal mucus (CVM) can provide a barrier that precludes HIV and other sexually transmitted virions from reaching target cells in the vaginal epithelium, thereby preventing or reducing infections. However, the barrier properties of CVM differ from woman to woman, and the causes of these variations are not yet well understood. Using high-resolution particle tracking of fluorescent HIV-1 pseudoviruses, we found that neither pH nor Nugent scores nor total lactic acid levels correlated significantly with virus trapping in unmodified CVM from diverse donors. Surprisingly, HIV-1 was generally trapped in CVM with relatively high concentrations of d-lactic acid and a Lactobacillus crispatus-dominant microbiota. In contrast, a substantial fraction of HIV-1 virions diffused rapidly through CVM with low concentrations of d-lactic acid that had a Lactobacillus iners-dominant microbiota or significant amounts of Gardnerella vaginalis, a bacterium associated with bacterial vaginosis. Our results demonstrate that the vaginal microbiota, including specific species of Lactobacillus, can alter the diffusional barrier properties of CVM against HIV and likely other sexually transmitted viruses and that these microbiota-associated changes may account in part for the elevated risks of HIV acquisition linked to bacterial vaginosis or intermediate vaginal microbiota. IMPORTANCE: Variations in the vaginal microbiota, especially shifts away from Lactobacillus-dominant microbiota, are associated with differential risks of acquiring HIV or other sexually transmitted infections. However, emerging evidence suggests that Lactobacillus iners frequently colonizes women with recurring bacterial vaginosis, raising the possibility that L. iners may not be as protective as other Lactobacillus species. Our study was designed to improve understanding of how the cervicovaginal mucus barrier against HIV may vary between women along with the vaginal microbiota and led to the finding that the vaginal microbiota, including specific species of Lactobacillus, can directly alter the diffusional barrier properties of cervicovaginal mucus. This work advances our understanding of the complex barrier properties of mucus and highlights the differential protective ability of different species of Lactobacillus, with Lactobacillus crispatus and possibly other species playing a key role in protection against HIV and other sexually transmitted infections. These findings could lead to the development of novel strategies to protect women against HIV.

Minimizing biases associated with tracking analysis of submicron particles in heterogeneous biological fluids.

Tracking the dynamic motion of individual nanoparticles or viruses offers quantitative insights into their real-time behavior and fate in different biological environments. Indeed, particle tracking is a powerful tool that has facilitated the development of drug carriers with enhanced penetration of mucus, brain tissues and other extracellular matrices. Nevertheless, heterogeneity is a hallmark of nanoparticle diffusion in such complex environments: identical particles can exhibit strongly hindered or unobstructed diffusion within microns of each other. The common practice in 2D particle tracking, namely analyzing all trackable particle traces with equal weighting, naturally biases towards rapidly diffusing sub-populations at shorter time scales. This in turn results in misrepresentation of particle behavior and a systematic underestimate of the time necessary for a population of nanoparticles to diffuse specific distances. We show here via both computational simulation and experimental data that this bias can be rigorously corrected by weighing the contribution by each particle trace on a 'frame-by-frame' basis. We believe this methodology presents an important step towards objective and accurate assessment of the heterogeneous transport behavior of submicron drug carriers and pathogens in biological environments.

Modeling of Virion Collisions in Cervicovaginal Mucus Reveals Limits on Agglutination as the Protective Mechanism of Secretory Immunoglobulin A.

Secretory immunoglobulin A (sIgA), a dimeric antibody found in high quantities in the gastrointestinal mucosa, is broadly associated with mucosal immune protection. A distinguishing feature of sIgA is its ability to crosslink pathogens, thereby creating pathogen/sIgA aggregates that are too large to traverse the dense matrix of mucin fibers in mucus layers overlying epithelial cells and consequently reducing infectivity. Here, we use modeling to investigate this mechanism of "immune exclusion" based on sIgA-mediated agglutination, in particular the potential use of sIgA to agglutinate HIV in cervicovaginal mucus (CVM) and prevent HIV transmission. Utilizing reported data on HIV diffusion in CVM and semen, we simulate HIV collision kinetics in physiologically-thick mucus layers-a necessary first step for sIgA-induced aggregation. We find that even at the median HIV load in semen of acutely infected individuals possessing high viral titers, over 99% of HIV virions will penetrate CVM and reach the vaginal epithelium without colliding with another virion. These findings imply that agglutination is unlikely to be the dominant mechanism of sIgA-mediated protection against HIV or other sexually transmitted pathogens. Rather, we surmise that agglutination is most effective against pathogens either present at exceedingly high concentrations or that possess motility mechanisms other than Brownian diffusion that significantly enhance encounter rates.