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.

Vaccination has been well recognised as a critically important tool in preventing infectious disease, yet incomplete immunisation coverage remains a major obstacle to achieving disease control and eradication. As medical products for global access, vaccines need to be safe, effective and inexpensive. In line with these goals, continuous improvements of vaccine delivery strategies are necessary to achieve the full potential of immunisation. Novel technologies related to vaccine delivery and route of administration, use of advanced adjuvants and controlled antigen release (single-dose immunisation) approaches are expected to contribute to improved coverage and patient compliance. This review discusses the application of micro- and nano-technologies in the alternative routes of vaccine administration (mucosal and cutaneous vaccination), oral vaccine delivery as well as vaccine encapsulation with the aim of controlled antigen release for single-dose vaccination.

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.

The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited. Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK (B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase (B. longum ssp. longum CCDM 372).

A semisynthetic strategy to obtain monophosphoryl lipid A derivatives equipped with clickable (azide, alkyne, double bond, or thiol precursor) moieties, starting from the native lipid A isolated from Escherichia coli, is presented. These lipid A derivatives can be conjugated with other interesting biomolecules, such as tumor-associated carbohydrate antigens (TACAs). In this way, the immunostimulant activity of monophosphoryl lipid A can significantly improve the immunogenicity of TACAs, thus opening access to potential self-adjuvant anticancer vaccine candidates. A monophosphoryl lipid A-Thomson-Friedenreich (TF) antigen conjugate was obtained to demonstrate the feasibility of this methodology, which stands as a valuable, rapid, and scalable alternative to the highly complex approaches of total synthesis recently reported to the same aim. A preliminary evaluation of the immunological activity of this conjugate as well as of other semisynthetic lipid A derivatives was also reported.

The classical definition of probiotics states that bacteria must be alive to be beneficial for human organism. However, recent reports show that inactivated bacteria or their effector molecules can also possess such properties. In this study, we investigated the physical and immunomodulatory properties of four Bifidobacterium strains in the heat-treated (HT) and untreated (UN) forms. We showed that temperature treatment of bacteria changes their size and charge, which affects their interaction with epithelial and immune cells. Based on the in vitro assays, we observed that all tested strains reduced the level of OVA-induced IL-4, IL-5, and IL-13 in the spleen culture of OVA-sensitized mice. We selected Bifidobacterium longum ssp. longum CCM 7952 (Bl 7952) for further analysis. In vivo experiments confirmed that untreated Bl 7952 exhibited allergy-reducing properties when administered intranasally to OVA-sensitized mice, which manifested in significant suppression of airway inflammation. Untreated Bl 7952 decreased local and systemic levels of Th2 related cytokines, OVA-specific IgE antibodies and simultaneously inhibited airway eosinophilia. In contrast, heat-treated Bl 7952 was only able to reduce IL-4 levels in the lungs and eosinophils in bronchoalveolar lavage, but increased neutrophil and macrophage numbers. We demonstrated that the viability status of Bl 7952 is a prerequisite for the beneficial effects of bacteria, and that heat treatment reduces but does not completely abolish these properties. Further research on bacterial effector molecules to elucidate the beneficial effects of probiotics in the prevention of allergic diseases is warranted.

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.

Clostridioides difficile (C. difficile) is an opportunistic anaerobic bacterium that causes severe diseases of the digestive tract of humans and animals. One of the possible methods of preventing C. difficile infection is to develop a vaccine. The most promising candidates for vaccine antigens are the proteins involved in the adhesion phenomena. Among them, the FliC and FliD are considered to be suitable candidates. In this paper, the FliC and FliD protein polypeptide epitopes were mapped in silico and by using PEPSCAN procedure. We identified four promising epitopes: 117QRMRTLS123, 205MSKAG209 of FliC and 226NKVAS230, 306TTKKPKD312 of FliD protein. We showed that 117QRMRTLS123 sequence is not only located in TLR5-binding and activating region, as previously shown, but forms an epitope recognized by C. difficile-infected patients’ antibodies. 205MSKAG209 is a C. difficile-unique, immunogenic sequence that forms an exposed epitope on the polymerized flagella structure which makes it a suitable vaccine antigen. 226NKVAS230 and 306TTKKPKD312 are well exposed and possess potential protective properties according to VaxiJen analysis. Our results open the possibility to use these epitopes as suitable anti-C. difficile vaccine antigens.