Aleksandra Inic-Kanada

Induction of APS after TTd Hyper‐Immunization has a Different Outcome in BALB/c and C57BL/6 Mice

Citation Zivkovic I, Stojanovic M, Petrusic V, Inic‐Kanada A, Dimitrijevic L. Induction of APS after TTd hyper‐immunization has a different outcome in BALB/c and C57BL/6 mice. Am J Reprod Immunol 2011; 65: 492–502

Induction of decreased fecundity by tetanus toxoid hyper-immunization in C57BL/6 mice depends on the applied adjuvant.

It has already been shown that tetanus toxoid (TTd) hyper-immunization is a suitable experimental method for creating the animal model of antiphospholipid syndrome (APS) in BALB/c mice. The severity of APS pathology in BALB/c mice mainly correlates to the affinity of anti-β2 glycoprotein I (β2GPI) antibodies. In this study we have investigated reproductive pathology induced in C57BL/6 mice by TTd hyper-immunization using a combination of different pretreatments (complete Freunds adjuvant or glycerol) and adjuvants (alhydrogel or glycerol). A decrease in fecundity was recorded in only C57BL/6 mice immunized with alhydrogel adjuvant, irrespective of the kind of applied pretreatment; it was associated with an increase in abundance of low affinity anti-β2GPI IgG antibodies and Th1 prevalence.

Quantification of the thaumatin-like kiwi allergen by a monoclonal antibody-based ELISA

Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.

The context of tetanus toxoid application influences the outcome of antigen-specific and self-directed humoral immune response

Results are presented concerning our attempts to create a suitable model system for studying the connection between microbial antigen (micAg), autoimmunity and autoimmune disease on the basis of hyper‐immunization and application of micAg in different contexts. Our research was focused on tetanus toxoid (TTd) as a model micAg. Non‐pretreated and complete Freunds adjuvant pretreated BALB/c mice were immunized with high doses of TTd mixed with glycerol or aluminum hydroxide as adjuvants. The main aims of the experiments were to evaluate the properties of induced humoral immune responses, evaluate the pathological potential of induced immune responses and determine possible correlations between the properties of a humoral immune response and its pathological potential. The production of TTd‐specific and self‐reactive β2‐glycoprotein I (β2‐GP I)‐specific antibodies (Abs) was detected in all groups but with specific, context‐related properties. Analysis of pregnancy‐related pathology (anti‐β2‐GP I Abs‐associated) showed differences in the pathological potential of the induced immune response. It was demonstrated that severity of pathology is positively correlated to the abundance of IgG that recognizes β2‐GP I adsorbed onto phosphatidylserine, and to IgG affinity. Furthermore, it was demonstrated that molecular mimicry, which results in generation of anti‐β2‐GP I Abs upon TTd immunization, is necessary but not sufficient for the development of pregnancy‐related pathology.

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.

Murine monoclonal antibody 26 raised against tetanus toxoid cross-reacts with beta2-glycoprotein I: its characteristics and role in molecular mimicry.

Problem  Studies on experimental antiphospholipid syndrome (APS) models proved that molecular mimicry between plasma protein β2‐glycoprotein I (β2GPI) and structure within micro‐organisms or their products, might be a cause for experimental APS. Considering the heterogeneity of polyclonal antiphospholipid antibodies (aPLs), it is important to define the precise characteristics of pathogenic aPLs. To avoid the influence of polyclonality and to further analyse the connection between molecular mimicry and APS, we produced monoclonal antibodies (MAbs) against tetanus toxoid (TTd) and tested their reactivity against β2GPI.

Development of Sandwich Enzyme‐Linked Immunosorbent Assay for Determination of Tetanus Toxoid Concentration

Abstract According to the recommendation of the World Health Organization (WHO), the use of an in vivo test for measuring of the potency of tetanus toxoid vaccine (TTdV) is still unavoidable, but the establishment of a convenient in vitro test would significantly improve the work in this field. A sandwich enzyme‐linked immunosorbent assay (sELISA) was developed for a rapid and sensitive quantification of tetanus toxoid (TTd). We produced four monoclonal antibodies (MAbs) designated 41, 51, 62, and 71 that reacted with TTd and recognized different antigenic determinants on TTd. We also used two of these antibodies for developing a sELISA, with MoAb 71 as an immobilized and MoAb 51 as a capture antibody. The measurement range of this assay was from 31–1000 ng/mL and the minimum detection limit for TTd was 31 ng/mL. This high sensitivity of this sELISA and its good reproducibility suggest that the developed method could be reliably used to estimate the concentration of TTd, which could be easily extrapolated to the estimation of vaccine potency.

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.

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen. Materials and methods BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 × LD50) of tetanus toxin. Results The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p<0.05). Conclusion Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively.

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.