Application of the 3r concept in the production of European antiviperinum on horses - multisite, low volumes immunization protocol and Elisa

During time, both professionals and general public became aware of the importance of animal welfare. This term not only covers endangered wild animal species, animals used in food industry, pets and experimental animals, but also animals used in production of biologics. The implementation of the 3R concept (Replacement, Reduction and Refinement) is especially important in this type of production. In this article, we describe for the first time the low dose, low volume and multi-site immunization protocol, as well as appropriate ELISA we developed for production of European anti-viper (V. ammodytes, long horned) antivenom in horses, which can help to significantly improve the welfare of the used animals

Monoklonsko antitelo 26 napravljeno na tetanus toksoidu reaguje i sa tetanus toksinom i β2-glikoproteinom I - karakteristike vezivanja in vitro i moguća primena

A murine monoclonal IgG1 antibody, marked as MAb26, specific for tetanus toxoid has been immunochemically characterized. By performing enzyme-linked immunosorbent assays (ELISAs) and western blot analyses, it was demonstrated that MAb26 reacted with tetanus toxoid, tetanus toxin and β2-glycoprotein I (β2GPI). According to the results, MAb26 recognized the sequential epitope on the tetanus heavy chain. The affinity constant, calculated from Scatchard plots of MAb26 binding to tetanus toxoid, was 1.145×108 M-1 and the measurement of the relative affinity of MAb26 by ELISA using thiocyanate elution showed a significantly higher affinity of MAb26 to the toxoid (p = 0.0012) in comparison to the toxin. Additionally, the reactivity of MAb26 toward the toxoid forms increased when the tetanus toxin was detoxified using 8 mM and higher formaldehyde concentrations. The similarity of the tetanus toxoid to several sera proteins, either at the level of its conformation (IL-1α) or at the level of peptide sequences (â2GPI, laminin) favors its role in autoimmunity by the mechanism of molecular mimicry. As the induction of an autoimmune disease is dependent on the breakdown of tolerance, which could be the result of an overt hyperstimulation, the control of the presence and concentration of self-reactive epitopes in vaccine preparations is a prerequisite. In this study, it was shown that MAb26 can: 1) discriminate between the tetanus toxin and different toxoid forms, which makes it a good candidate for antibody control during vaccine preparation; 2) due to its cross-reactivity with β2GPI, it could provide information on the presence of a potentially dangerous sequential epitope expressed at the protein surface.Ovaj rad opisuje imunohemijsku karakterizaciju mišjeg IgG1 monoklonskog antitela označenog kao MAt26. Enzimskim imunosorbentnim testom (ELISA) i Western blot analizom je pokazano da MAt26 reaguje sa tetanus toksoidom, tetanus toksinom i β2-glikoproteinom I (β2GPI). Prema našim rezultatima, MAt26 prepoznaje sekvencioni epitop na teškom lancu molekula tetanusa. Konstanta afiniteta MAt26 za tetanus toksoid, izračunata na osnovu Skačardovog dijagrama, je 1,145×108 M-1. Na osnovu elucije tiocijanatom, korišćene za određivanje relativnog afiniteta MAt26 za tetanus toksin i tetanus toksoid, postupkom baziranim na ELISA-i, pokazan je znatno veći (p = 0,0012) afinitet MAt26 ka toksoidnoj formi. Takođe, reaktivnost MAt26 ka toksoidnoj formi rasla je sa porastom koncentracije formaldehida, počevši od 8 mM, korišćenog u procesu detoksifikacije. Sličnost tetanus toksoida sa različitim serumskim proteinima na nivou konformacije i/ili peptidnih sekvencija (β2GPI, laminin) ukazuje na njegovu potencijalnu ulogu u indukciji autoimunosti mehanizmom molekulske mimikrije. Budući da nastanak autoimunske bolesti podrazumeva narušavanje tolerancije, na primer, prekomernom stimulacijom imunskog sistema, kontrola prisustva i koncentracije sebi sličnih epitopa se nameće kao neophodna. U ovom radu je pokazano da: 1) MAt26 može da pravi razliku između tetanus toksina i različitih toksoidnih formi što ga čini potencijalno dobrim antitelom koje bi se koristilo u kontroli tokom proizvodnje vakcina; 2) zahvaljujući unakrsnoj reaktivnosti sa β2GPI, MAt26 može da pruži informacije o prisustvu potencijalno opasnih epitopa na površini proteina

Karakterizacija Intor:Swiss soja albino miševa donetog u Institut za virusologiju, vakcine i serume - Torlak početkom XX veka

The Institute of Virology, Vaccines and Sera Torlak was established in 1927, while the first vaccine was produced in the Institute in 1930. Vaccines production implies using experimental animals, including mice, in in-process controls. The laboratory mice which have been in use in Torlak Institute from the very beginning belong to Swiss albino outbred stock. This stock, which has been in use for more than 80 years contains a large number of mice maintained at all times, was recently named Intor:Swiss. Biological characteristics of Intor:Swiss stock, are presented in this paper for the first time. Taking into account the presented characteristics, the Institute Torlak's Swiss mice are suitable for use in pharmaceutical studies, vaccine development research and basic research, as well as in toxicological studies. The publication of data on the Intor:Swiss mice represents a contribution to the international scientific community, since it offers the possibility for obtaining an additional outbred mouse stock for research.Institut za Virusologiju, vakcine i serume Torlak, osnovan je 1927., a prva vakcina u Institutu proizvedena je 1930. Proizvodnja vakcina je složen proces koji između ostalog podrazumeva i korišćenje eksperimentalnih životinja u kontroli samog procesa. Laboratorijski miševi koji su od samog početka bili u upotrebi u Institutu Torlak, pripadaju Swiss albino outbred soju. Ova kolonija je u upotrebi više od 80 godina i sve vreme se sastoji od velikog broja jedinki što omogućava očuvanje genetske raznolikosti, pa samim tim i outbred karakteristika. Ovi miševi su odnedavno registrovani pod imenom Intor:Swiss, i njihove biološke osobine su u ovom radu prikazane po prvi put. Swiss miševi Instituta Torlak pogodni su za upotrebu u farmaceutskim studijama, za razvojno istraživanje vakcina, osnovna istraživanja i toksikološka ispitivanja. Zbog svega navedenog Intor:Swiss miševi predstavljaju još jedan pogodan animalni model za ispitivanje lekova i vakcina

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity

Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice

One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein beta(2)GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-beta(2)GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-beta(2)GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies

Optimization and Validation of ELISA for Pre-Clinical Trials of Influenza Vaccine

Testing of every new vaccine involves investigation of its immunogenicity, which is based on monitoring its ability to induce specific antibodies in animals. The fastest and most sensitive method used for this purpose is enzyme-linked immunosorbent assay (ELISA). However, commercial ELISA kits with whole influenza virus antigens are not available on the market, and it is therefore essential to establish an adequate assay for testing influenza virus-specific antibodies. We developed ELISA with whole influenza virus strains for the season 2011/2012 as antigens and validated it by checking its specificity, accuracy, linearity, range, precision, and sensitivity. The results show that we developed high-quality ELISA that can be used to test immunogenicity of newly produced seasonal or pandemic vaccines in mice. The pre-existence of validated ELISA enables shortening the time from the process of vaccine production to its use in patients, which is particularly important in the case of a pandemic

Changes in Composition of IgM Polymers in Patients Suffering from Recurrent Urinary Bacterial Infections after Bacterial Immunization Treatment

IgM is the first antibody produced during the immune response to infection or immunization, and it can be secreted as pentamer (containing a small polypeptide, termed as J chain) or hexamer (lacking J chain). In this paper we have analyzed structural characteristics (by electrophoresis and immunoblot) and anti-bacterial specificity (by enzyme-linked immunosorbent assay) of IgM antibodies purified from female patients suffering from recurrent bacterial infections of lower urinary tract and therapeutically immunized with the mixture of heat-inactivated uropathogenic bacteria. We report on changes in the composition of IgM polymers in tested patients. The immunization induced the raise of the levels of hexamers in patients that did not respond to immunization (non-responders), while the IgM polymers remained on the pentameric level in immunization dependent responders. We propose that the composition of IgM polymers could influence the immunization outcome and should be taken in count regarding the treatment of recurrent bacterial infections

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

Problem The antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by vascular thrombosis and/or pregnancy complications (lower fecundity and lower litter size), as well as by an increase in anti-beta(2) glycoprotein I (beta(2)GPI)-specific autoantibody titer. We have investigated how the genetic background of the immune system [T helper (Th) prevalence] and the type of animal model of APS influence the induced pathology. Method of Study Antiphospholipid syndrome induced by tetanus toxoid (TTd) hyper-immunization and by intravenous application of monoclonal anti-beta(2)GPI-specific antibody 26 was compared in C57BL/6 (Th1 prone) and BALB/c (Th2 prone) mice. Results Tetanus toxoid hyper-immunization of BALB/c mice led to reduction in fertility, but in C57BL/6 mice a decrease in fecundity occurred. In both cases, pathology was caused by anti-beta(2)GPI antibodies, the production of which was adjuvant and strain dependent. Conclusion We conclude that TTd immunization and i.v. application of monoclonal antibody 26 induced the same reproductive pathology and that the type of pathology is strain dependent

Vaccine model of antiphospholipid syndrome induced by tetanus vaccine

Successful induction of antiphospholipid syndrome (APS) in two different non-autoimmune prone mouse strains, BALB/c and C57BL/6, was achieved by tetanus toxoid (TTd) hyperimmunization using different adjuvants (glycerol or aluminium hydroxide), and different adjuvant pretreatments (glycerol or Complete Freund's Adjuvant (CFA)). APS had different manifestations of reproductive pathology in BALB/c and C57BL/6 mice: fetal resorption (as a consequence of extreme T-cell activation obtained in the course of pretreatment), and lowering of fecundity (as a consequence of polyclonal B-cell stimulation), respectively. In BALB/c mice fetal resorption coincided with glycerol and CFA pretreatments, while in C57BL/6 mice lowering of fecundity was most obvious in CFA-pretreated mice immunized with TTd in aluminium hydroxide. Both molecular mimicry and polyclonal B-cell activation occur in APS induction, with molecular mimicry effects being dominant in BALB/c mice, and polyclonal cell activation being dominant in C57BL/6 mice. Confirmation of molecular mimicry effects, which in the condition of T-cell stimulation generated fetal resorptions in the BALB/c strain, was achieved by passive infusion of monoclonal antibody (MoAb) T-26 specific for TTd and anti-beta(2)-glycoprotein I obtained after TTd hyperimunization. High polyclonal B-cell activation in C57BL/6 mice prevented fetal resorption but induced fecundity lowering, as was the case in passive administration of MoAb T-26 in this mouse strain. Passive infusion of anti-idiotypic MoAb Y7 into C57BL/6 mice induced fetal resorptions and confirmed the above suggestion on the protective role of polyclonal B-cell stimulation in fetal resorptions. Lupus (2012) 21, 195-202