Effective mucosal vaccines - opportunities and challenges / Efektywne szczepionki sluzówkowe ­ mozliwosci i wyzwania.

Most pathogens enter the body through the surfaces of the mucous membranes, e.g. the nose or the intestines. The mucosal immune response is essential for the effective elimination of invading pathogens. Unfortunately, most vaccines which are administered intramuscularly by injection do not induce an adequate protective immune response on mucous membranes. For example, after intramuscular injection, the level of secretory IgA antibodies is low and often insufficient to successfully combat the pathogen. On the other hand, mucosal-induced immunity produces a long-lasting effect in the form of a local and systemic response to the pathogen. Moreover, the administration of such vaccines does not generate hazardous medical waste and their application does not require the presence of qualified medical personnel. Therefore, intensive research into vaccines administered via the mucosal route is ongoing. An obstacle in the development of mucosal vaccines is the natural defense mechanisms of the mucosa, the overcoming of which requires the use of specialized adjuvants. Currently, there are no such formulations on the market.

[Selected aspects of Clostridium difficile infection]. / Wybrane aspekty zakazen Clostridium difficile.

Clostridium difficile pathogen is a cause of the most frequent nosocomial infection, which is antibiotic-associated diarrhea. Antibiotic treatment causes disruption of the microbiome balance, which makes the gut a friendly environment for the pathogen. It leads to pseudomembranous colitis, toxic megacolon and even death. Clostridium difficile infection (CDI) is particularly dangerous to elderly patients, leading to the highest mortality rate. C. difficile is equipped with many virulence factors such as toxin A and B, binary toxin CDT, flagellum, S-layer proteins, Cwp66 and GroEL proteins, protease Cwp84, fibronectin-binding protein and the ability to form biofilm and spores. Problems with anti-CDI therapy prompt researchers and clinicians to seek alternative ways of therapy. Identification of immunological epitopes in outer layer proteins and the use of them as antigens for anti-CDI vaccines would be a rational approach to prevent the disease, but unfortunately such vaccines are not available yet. In this article we review the course of the disease, virulence and risk factors. We summarize briefly epidemiological data and the latest achievements in CDI treatment.

OT-II TCR transgenic mice fail to produce anti-ovalbumin antibodies upon vaccination.

OT-II mice were evaluated as a transgenic strain-specific model to assess T-cell help for B-cell responses. OT-II CD4(+) T-cells express transgenic OVA-specific αß-TCRs. This high frequency of antigen-specific helper T-lymphocytes may augment induction of B-cell responses. Unexpectedly, OT-II mice did not produce OVA-specific antibodies after intranasal immunization. However, B-cells expressed normal antigen-presenting function in vitro for activation of OVA-specific T-cell responses. These OT-II T-cell responses produced a Th1-type cytokine profile with significantly reduced Th2 or Th17 responses. These data suggest that OT-II B-cells are not defective as APCs, however, downstream antibody responses are abrogated in this transgenic strain.

Silicone Oil-Based Nanoadjuvants as Candidates for a New Formulation of Intranasal Vaccines.

Many conventional vaccines are administered via a needle injection, while most pathogens primarily invade the host via mucosal surfaces. Moreover, protective IgA antibodies are insufficiently induced by parenteral vaccines. Mucosal immunity induces both local and systemic response to pathogens and typically lasts for long periods of time. Therefore, vaccination via mucosal routes has been increasingly explored. However, mucosal vaccines require potent adjuvants to become efficacious. Despite many efforts to develop safe and robust adjuvants for mucosal vaccines, only a few have been approved for use in human formulations. The aim of our study was to design, develop and characterize new silicone oil-based nanoadjuvant candidates for intranasal vaccines with potential to become mucosal adjuvants. We have developed an array of nanoadjuvant candidates (NACs), based on well-defined ingredients. NAC1, 2 and 3 are based on silicone oil, but differ in the used detergents and organic solvents, which results in variations in their droplet size and zeta potential. NACs' cytotoxicity, Tumor Necrosis Factor α (TNF-α) induction and their effect on antigen engulfment by immune cells were tested in vitro. Adjuvant properties of NACs were verified by intranasal vaccination of mice together with ovalbumin (OVA). NACs show remarkable stability and do not require any special storage conditions. They exhibit bio-adhesiveness and influence the degree of model protein engulfment by epithelial cells. Moreover, they induce high specific anti-OVA IgG antibody titers after two intranasal administrations. Nanoadjuvant candidates composed of silicone oil and cationic detergents are stable, exhibit remarkable adjuvant properties and can be used as adjuvants for intranasal immunization.

Targeting the efficacy of a dendrimer-based nanotherapeutic in heterogeneous xenograft tumors in vivo.

Our earlier studies have shown the in vitro and in vivo targeting of a generation 5 (G5) dendrimer-based multifunctional conjugate that contained folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug. To clinically apply the synthesized G5-FA-MTX nanotherapeutic, it is important that the anticancer conjugate elicits cytotoxicity specifically and consistently. Toward this objective, we evaluated the large-scale synthesis of a G5-FA-MTX conjugate (Lot # 123-34) for its cytotoxic potential and specificity in vitro and in vivo. The cytotoxicity and specificity were tested by using a coculture assay in which FA receptor-expressing and nonexpressing cells (KB and SK-BR-3 cells, respectively) were cultured together and preferential killing was examined. The in-vitro data were compared with the in-vivo data obtained from a heterogeneous xenograft tumor model. The animal model of the artificial heterogeneous xenograft tumor showed that the nanotherapeutic was preferentially cytotoxic to KB cells.

Efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model.

Mutations of influenza virus increase concerns of worldwide epidemics resulting from the newly emergent strains. Current influenza vaccines are inefficient and require annual vaccinations. W805EC adjuvant is an oil-in-water emulsion composed of nanodroplets with an average diameter of approximately 400 nm. The nanoemulsion adjuvant has been used successfully to stimulate the immune response when mixed with several other antigens in animal models. In this study, W805EC nanoemulsion adjuvant activity was evaluated using nasal influenza vaccination in a murine model. Five to twenty percent W805EC adjuvant was used to inactivate influenza A/Puerto Rico/8/34/05 (H1N1). Mice immunized with the nanoemulsion adjuvanted influenza virus intranasally showed a robust specific humoral immune response as demonstrated using ELISA and HAI assays. Serum HAI titers were more than 104 following two vaccinations. Vaccinated mice were also protected against challenge with an LD80 of live influenza virus. Splenocytes from vaccinated mice were assayed for cytokine production following virus stimulation. The cytokine profile demonstrated a robust cellular immune response with enhanced Th1 and Th17 immunity that provided balanced immunity against both intracellular and extracellular forms of the virus. Additionally, the vaccine preparations showed minimal protein degradation but remained potent when stored at 4°C for up to three months. This work demonstrates that W805EC nanoemulsion adjuvant can effectively enhance the immunogenicity of influenza hemagglutinin antigen. The nanoemulsion adjuvant can result in antigen sparing and cross-protection. The potential exists for a nasally administered influenza vaccine that may require little or no refrigerated storage.

The Bioinformatic and In Vitro Studies of Clostridioides Difficile Aminopeptidase M24 Revealed the Immunoreactive KKGIK Peptide.

Clostridioides difficile (CD) is a Gram-positive pathogen responsible for CD-associated disease (CDAD), which is characterized by symptoms ranging from mild diarrhea to pseudomembranous colitis. This work is an attempt to respond to the need of novel methods for CD infection (CDI) prevention, since the number of CDI cases is still rising. A bioinformatics approach was applied to design twenty-one peptides consisting of in silico predicted linear B-cell and T-cell epitopes of aminopeptidase M24 from CD. These peptides were mapped for epitopes exploiting PEPSCAN procedure and using sera obtained from CD infected patients, umbilical cord blood, and healthy volunteers. Two new CD epitopes, 131KKGIK135 and 184KGTSTHVIT192, were identified and characterized. Immunoreactivity of the synthetic biotinylated 131KKGIK135 epitope was significantly higher compared to 184KGTSTHVIT192 epitope in Enzyme-Linked Immunosorbent Assay (ELISA) with umbilical cord blood and CDI patients' sera. Hereafter, the conjugate of bovine serum albumin and epitope 131KKGIK135 was evaluated in vitro on lung epithelial cell line. In vitro, a significant induction of IL-6 by conjugate was observed, thereby we postulate that this new 131KKGIK135 epitope possesses immunostimulating properties suggesting possibility of its use in a vaccine against Clostridioides difficile.

Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood.

Clostridioides difficile (CD) cause a severe diarrhea which can lead to pseudomembranous colitis and even patient death. CD infection (CDI) is connected mainly with changes in intestinal microbiota as a consequence of antibiotic treatment. The growing resistance to antibiotics, justifies the search for new methods of combating CD. Despite of ongoing research on the immunity against the pathogen, there is still lack of any reliable vaccine. Most recently, Cwp22, that is a cross-linking enzyme involved in the production of CD peptidoglycan, seems to be a promising target to prevent CDI in high-risk patients. In this paper, the Cwp22 protein polypeptide-specific epitopes were mapped in silico and using PEPSCAN procedure. They were recognized not only by antibodies from CDI patients' but also by umbilical cord blood sera. We identified three epitopes 54EFRVAT59, 201KVNGKM206 and 268WQEKNGKKYY277 of Cwp22 protein. Since Cwp22 protein has key functionality and the described above epitopes are also recognized by umbilical cord blood serum, we postulate that they could have important protective properties. In this paper, we propose Cwp22 protein as a good antigen candidate for CDI preventive vaccine. Our results open the possibility to use 54EFRVAT59, 201KVNGKM206 and 268WQEKNGKKYY277, epitopes as suitable anti-CD vaccine antigens.

Sensitive molecular binding assay using a photonic crystal structure in total internal reflection.

A novel optical sensor for label-free biomolecular binding assay using a one-dimensional photonic crystal in a total-internal-reflection geometry is proposed and demonstrated. The simple configuration provides a narrow optical resonance to enable sensitive measurements of molecular binding, and at the same time employs an open interface to enable real-time measurements of binding dynamics. Ultrathin aminopropyltriethoxysilane/ glutaraldehyde films adsorbed on the interface were detected by measuring the spectral shift of the photonic crystal resonance and the intensity ratio change in a differential reflectance measurement. A detection limit of 6 x 10(-5) nm for molecular layer thickness was obtained, which corresponds to a detection limit for analyte adsorption of 0.06 pg/mm(2) or a refractive index resolution of 3 x 10(-8) RIU; this represents a significant improvement relative to state-of-the-art surface-plasmon-resonance-based systems.

Extended cavity laser enhanced two-photon flow cytometry.

We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP)-expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels.

Quantitative two-photon flow cytometry--in vitro and in vivo.

Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.

In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry.

To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.

Comparison of Commensal Escherichia coli Isolates from Adults and Young Children in Lubuskie Province, Poland: Virulence Potential, Phylogeny and Antimicrobial Resistance.

Commensal Escherichia coli population is a dynamic structure which may be important in the pathogenesis of extraintestinal infections. The aim of this study was the comparison of genetic diversity of commensal E. coli isolates from two age group-adults and young children. E. coli strains were isolated on MacConkey agar and identified by biochemical tests. Determination of four major phylogenetic groups, identification of virulence genes and antimicrobial resistance determinants were performed by using multiplex or simplex PCR. Phenotypic analysis of resistance was based on disc-diffusion method. The prevalence of virulence genes was significantly higher among isolates from adults than from young children. Phylogroup B2 predominated among E. coli from adults, whereas phylogroup A was the most common in isolates from young children. The analyses of antimicrobial resistance revealed that resistance to at least one antimicrobial agent and multidrug-resistance were detected significantly more frequent in the isolates from adults than from young children. This study documented that the commensal E. coli isolates from adults showed greater genetic diversity than from young children and constitutes a substantial reservoir of the virulence genes typical for extraintestinal pathogenic E. coli.

Epitopes identified in GAPDH from Clostridium difficile recognized as common antigens with potential autoimmunizing properties.

Clostridium difficile (CD) infections are a growing threat due to the strain resistance to antibiotic treatment and the emergence of hypervirulent strains. One solution to this problem is the search for new vaccine antigens, preferably surface-localized that will be recognized by antibodies at an early stage of colonization. The purpose of the study was to assess the usefulness of novel immunoreactive surface proteins (epitopes) as potential vaccine antigens. Such approach might be tough to pursue since pathogens have acquired strategies to subvert adaptive immune response to produce humoral response against non-essential proteins for their survival. In this study CD surface proteins were isolated, immunoreactive proteins identified and mapped to select potential epitopes. The results of the study exclude the use of CD glyceraldehyde 3-phosphate dehydrogenase as a vaccine antigen, especially as a whole protein. Sequences P9 (201AAGNIVPNTTGAAKAI218) and P10 (224KGKLDGAAQRVPVVTG241) recognized by patients sera are conserved and widespread among CD strains. They show cross-reactivity with sera of people suffering from other bacterial infections and are recognized by sera of autoimmune disease patients. Our study documents that special care in analyzing the sequence of new epitope should be taken to avoid side effects prior to consider it as a vaccine antigen.

Targeting and inhibition of cell growth by an engineered dendritic nanodevice.

The cellular uptake and cytotoxicity of an engineered multifunctional dendritic nanodevice containing folic acid (FA) as the targeting molecule, methotrexate (MTX) as the chemotherapeutic drug, and fluorescein (FI) as the detecting agent were studied in vitro. FI and FA were conjugated to the generation 5 poly(amidoamine) (G5) dendrimer carrier through a thiourea and amide linkage and MTX was conjugated through an ester linkage to the carrier to generate the trifunctional dendritic device, G5-FI-FA-MTX. This trifunctional dendrimer-drug conjugate bound to FA receptor-expressing KB cells in a dose-dependent and saturable manner. Confocal microscopic analysis demonstrated cellular internalization of the conjugate. G5-FI-FA-MTX induced a time- and dose-dependent inhibition of cell growth in KB cells. The targeted dendrimer conjugates G5-FI-FA-MTX and G5-FA-MTX inhibited cell growth in KB cells, whereas the nontargeted G5-MTX failed to induce growth inhibition. These studies show the potential of G5-FI-FA-MTX or G5-FA-MTX for targeting and growth suppression of tumor cells that overexpress FA-receptors.

Tumor angiogenic vasculature targeting with PAMAM dendrimer-RGD conjugates.

PAMAM dendrimer-RGD-4C peptide conjugate was synthesized and in vitro targeting efficacy to integrin receptor expressing cells was studied by flow cytometry and confocal microscopy.

Nanoemulsion-based mucosal adjuvant induces apoptosis in human epithelial cells.

Nanoemulsions (NEs) are adjuvants that enhance antigen penetration of the nasal mucosa, increase cellular uptake of antigens by both epithelial and dendritic cells, and promote the migration of antigen-loaded dendritic cells to regional lymph nodes within 24-h of vaccine administration. The objective of this study was to elucidate cell death caused by W805EC NE and identify caspases and genes associated with death pathways. Consistent with this aim, we show that exposure of human epithelial cells (EC), both RPMI 2650 and FaDu, to NE results in the activation of caspases (1, 3/7, 6, 8, and 9) and the expression of genes involved in apoptotic as well as authophagy and necrosis pathways. Interestingly, the NE activates caspase 8 which promotes "immunogenic apoptosis". The rescue assay was employed to investigate the fate of RPMI 2650 cells treated with W805EC NE. After four-hour treatment with as little as 0.03% of NE no cells were rescued at 72h. Remarkably, immediately after four-hour treatment, the cells morphologically resembled untreated cells and most of the cells were alive. Altogether, these results suggest that NE induces death of human ECs through multiple pathways. Epithelial cell death caused by W805EC may have further implications on antigen uptake, processing, and presentation by DC's.

Formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants.

Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications.

Intranasal immunization with W 80 5EC adjuvanted recombinant RSV rF-ptn enhances clearance of respiratory syncytial virus in a mouse model.

Respiratory Syncytial Virus (RSV) is a ubiquitous virus that infects almost all people by age two and is a major source of respiratory illness in infants, the elderly and others with compromised immune systems. Currently there is no available vaccine. Prior efforts using formalin-inactivated RSV (FI-RSV) were associated with enhanced respiratory disease upon viral exposure following clinical vaccine trials. Several researchers and pharmaceutical companies have utilized vector-associated live attenuated RSV vaccines in pre-clinical and clinical studies. Another attractive approach, however, is a subunit vaccine which would be easier to produce and quality control. Our group has previously demonstrated in a murine model of infection that intranasal immunization with nanoemulsion-inactivated and adjuvanted RSV induces humoral and cellular immune responses, resulting in protection against RSV infection. The present studies characterize the immune responses elicited by intranasal RSV F protein adjuvanted with nanoemulsion. Intranasal application of nanoemulsion adjuvanted F protein induced a rapid and robust systemic and mucosal antibody response, as well as protection against subsequent RSV challenge. Importantly, RSV challenge in immunized animals did not elicit airway hyper-reactivity, a Th2-skewed immune response or immunopathology associated with hypersensitivity reactions with formalin-inactivated vaccine. These results suggest that RSV F protein adjuvanted with nanoemulsion may be a good mucosal vaccine candidate. Formulating RSV F protein in nanoemulsion creates a well-defined and well-controlled vaccine that can be delivered intranasally to induce T cell mediated immunity without inducing enhanced disease associated with the mouse model of FI-RSV vaccination and infection.

Nanoemulsion nasal adjuvant W805EC induces dendritic cell engulfment of antigen-primed epithelial cells.

Nanoemulsions are adjuvants that enhance antigen penetration in the nasal mucosa, increase cellular uptake of antigens by both epithelial dendritic cells, and promote migration of antigen-loaded dendritic cells to regional lymph nodes within a day of vaccine administration. The objective of this study was to determine whether the W(80)5EC nanoemulsion adjuvant enhances immune response not only by direct uptake of antigen by dendritic cells, but also indirectly, by phagocytosis of antigen-primed, apoptotic, epithelial cells. Consistent with this, we show that exposure of both epithelial cells (TC-1s) and dendritic cells (JAWS II or bone marrow derived dendritic cells (BMDCs)) to nanoemulsion exhibited augmented antigen uptake in cell culture. TC-1 cells subsequently underwent G(2)/M cell cycle arrest and apoptosis, and when co-cultured with JAWS II or BMDCs were rapidly engulfed by the dendritic cells, which responded by up-regulating dendritic cell maturation marker CD86. Altogether these results suggest that the effectiveness of nanoemulsions as adjuvants stems, at least in part, from the engulfment of antigen-loaded epithelial cells, leading to enhanced antigen processing and a strong and balanced mucosal and systemic immune response.