Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers.

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.

Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

In vitro and in vivo uptake study of Escherichia coli Nissle 1917 bacterial ghosts: cell-based delivery system to target ocular surface diseases.

PURPOSE: For the successful topical administration of drugs or vaccines to treat ocular surface diseases, efficient and well-tolerated delivery systems/carriers for conjunctival delivery are crucial in the development of new treatment strategies. The present study investigated the efficiency of internalization of bacterial ghosts (BGs) produced from probiotic Escherichia coli Nissle 1917 (EcN) by human conjunctival epithelial (HCjE) cell line, the EcN BGs cytotoxicity for HCjE cells, and in vivo uptake of EcN BGs by conjunctival guinea pig epithelial cells. METHODS: The uptake of EcN BGs by HCjE cells was analyzed by laser scanning microscopy and flow cytometry. Immunohistochemistry was used to localize the EcN BGs in the guinea pig conjunctival tissue. Cytotoxicity of EcN BGs on HCjE cells was evaluated by measurement of LDH. RESULTS: Laser scanning microscopy and flow cytometry revealed that EcN BGs internalization by HCjE cells was time- and dose dependent. No cytotoxic effect on HCjE cells was observed after EcN BGs inoculation for 30 and 120 minutes, as well as 24 hours. In addition, the uptake of EcN BGs was detected in the conjunctival cells after in vivo administration of EcN BGs into the eye of the guinea pig. CONCLUSIONS: The findings that EcN BGs are nontoxic and effectively internalized in vitro by human and in vivo by guinea pig conjunctival cells comprise an important contribution to the future use of BGs as a system for conjunctival delivery of drugs and vaccines, either to treat or prevent ocular surface diseases.

Comparison of ophthalmic sponges and extraction buffers for quantifying cytokine profiles in tears using Luminex technology.

PURPOSE: Evaluating cytokine profiles in tears could shed light on the pathogenesis of various ocular surface diseases. When collecting tears with the methods currently available, it is often not possible to avoid the tear reflex, which may give a different cytokine profile compared to basal tears. More importantly, tear collection with glass capillaries, the most widely used method for taking samples and the best method for avoiding tear reflex, is impractical for remote area field studies because it is tedious and time-consuming for health workers, who cannot collect tears from a large number of patients with this method in one day. Furthermore, this method is uncomfortable for anxious patients and children. Thus, tears are frequently collected using ophthalmic sponges. These sponges have the advantage that they are well tolerated by the patient, especially children, and enable standardization of the tear collection volume. The aim of this study was to compare various ophthalmic sponges and extraction buffers to optimize the tear collection method for field studies for subsequent quantification of cytokines in tears using the Luminex technology. METHODS: Three ophthalmic sponges, Merocel, Pro-ophta, and Weck-Cel, were tested. Sponges were presoaked with 25 cytokines/chemokines of known concentrations and eluted with seven different extraction buffers (EX1-EX7). To assess possible interference in the assay from the sponges, two standard curves were prepared in parallel: 1) cytokines of known concentrations with the extraction buffers and 2) cytokines of known concentrations loaded onto the sponges with the extraction buffers. Subsequently, a clinical assessment of the chosen sponge-buffer combination was performed with tears collected from four healthy subjects using 1) aspiration and 2) sponges. To quantify cytokine/chemokine recovery and the concentration in the tears, a 25-plex Cytokine Panel and the Luminex xMap were used. This platform enables simultaneous measurement of proinflammatory cytokines, Th1/Th2 distinguishing cytokines, nonspecific acting cytokines, and chemokines. RESULTS: WE DEMONSTRATED THE FOLLOWING: (i) 25 cytokines/chemokines expressed highly variable interactions with buffers and matrices. Several buffers enabled recovery of similar cytokine values (regulated and normal T cell expressed and secreted [RANTES], interleukin [IL]-13, IL-6, IL-8, IL-2R, and granulocyte-macrophage colony-stimulating factor [GM-CSF]); others were highly variable (monocyte chemotactic protein-1 [MCP-1], monokine induced by interferon-gamma [MIG], IL-1ß, IL-4, IL-7, and eotaxin). (ii) Various extraction buffers displayed significantly different recovery rates on the same sponge for the same cytokine/chemokine. (iii) The highest recovery rates were obtained with the Merocel ophthalmic sponge except for tumor necrosis factor-α: the Weck-Cel ophthalmic sponge showed the best results, either with cytokine standards loaded onto sponges or with tears collected from the inner canthus of the eye, using the sponge. (iv) IL-5, IL-10, and interferon-α were not detected in any tear sample from four normal human subjects. Twenty-two cytokines/chemokines that we detected were extracted from the Merocel sponge to a satisfactory recovery percentage. The recovery of IL-7 was significantly lower in the extracted Merocel sponge compared to the diluted tear samples. The cytokine/chemokine extraction from tears showed the same pattern of extraction that we observed for extracting the standards. CONCLUSIONS: Simultaneous measurement of various cytokines using ophthalmic sponges yielded diverse results for various cytokines as the level of extraction differs noticeably for certain cytokines. A second set of controls (standard curves "with sponges") should be used to delineate the extent of extraction for each cytokine to be analyzed. Many cytokines/chemokines were detected in tear samples collected with the Merocel sponge, including many that have been implicated in ocular surface disease. Luminex detection of cytokine/chemokine profiles of tears collected with Merocel sponges and extracted with buffer EX1 may be useful in clinical studies, for example, to assess cytokine profiles evaluation in ocular surface diseases.