Bacterial vaginosis (BV) affects almost a quarter of US women, making it a condition of major public health relevance. Key questions remain regarding the etiology of BV, mechanisms for its association with poor reproductive health outcomes, and reasons for high rates of treatment failure. New model systems are required to answer these remaining questions, elucidate the complex host-microbe and microbe-microbe interactions, and develop new, effective interventions. In this review, we cover the strengths and limitations of in vitro and in vivo model systems to study these complex intercellular interactions. Furthermore, we discuss advancements needed to maximize the translational utility of the model systems. As no single model can recapitulate all of the complex physiological and environmental conditions of the human vaginal microenvironment, we conclude that combinatorial approaches using in vitro and in vivo model systems will be required to address the remaining fundamental questions surrounding the enigma that is BV.
Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.
Our understanding of the mechanisms that regulate tissue-specific mucosal defense can be limited by the lack of appropriate human in vitro models. The endocervix lies between the microbe-rich vaginal cavity and the relatively sterile endometrium and is a major portal of entry for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, human immunodeficiency virus (HIV), and herpes simplex virus (HSV) infection in women. The endocervix is lined with a simple epithelium, and these cells produce mucus, which plays a key role in immune defense and reproduction. Here we describe the development of a human three-dimensional endocervical epithelial cell model generated by rotating wall vessel bioreactor technology. The model is composed of cellular aggregates that recapitulate major structural and barrier properties essential for the function and protection of the endocervix, including junctional complexes, microvilli, innate immune receptors, antimicrobial peptides, and mucins, the major structural component of mucus. Using this model, we also report, for the first time, that the membrane-associated mucin genes MUC1, MUC4, and MUC16 are differentially regulated in these aggregates by different bacterial and viral products. Differential induction of antimicrobial peptides was also observed with these products. Together these data define unique and flexible innate endocervical immune signatures that follow exposure to microbial products and that likely play a critical role in the outcome of pathogen challenge at this site.
Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.
Noroviruses (NoV) cause the great majority of epidemic nonbacterial gastroenteritis in humans. Expression of the capsid protein in recombinant systems, including insect and plant cells, yields assembly of virus-like particles (VLPs) that mimic the antigenic structure of authentic virions, and are relatively acid- and heat-stable. Norwalk virus (NV), the prototype NoV, has been studied extensively, and Norwalk virus-like particles (NVLPs) produced in insect cells and plants are immunogenic in mice and humans when delivered orally, stimulating the production of systemic and mucosal anti-NV antibodies. NVLPs are also highly immunogenic when delivered intranasally, provoking antibodies at levels similar to orally delivered VLP at much lower doses. Oral and nasal delivery of NVLPs efficiently produces antibodies at distal mucosal sites, which suggests that NVLPs could be used to deliver heterologous peptide antigens by production of genetic fusion chimeric capsid proteins. Examination of norovirus VLP surface structures and receptor binding motifs facilitates identification of potential sites for insertion of foreign peptides that will minimally affect the efficiency of VLP assembly and receptor binding. Thus, there is strong potential to use norovirus VLPs as vaccine-delivery vehicles.
