Selection of Candidate Monoclonal Antibodies for Therapy of Botulinum Toxin Type A Intoxications.

Botulism is one of the most serious food intoxications, manifesting as prolonged paralytic conditions. This disease is usually the result of the consumption of poor quality canned or smoked foods, so the inhabitants of many countries of the world are exposed to the risk of this kind of poisoning every year. In view of the severity of poisonings caused by botulinum neurotoxins, monoclonal antibodies (mAbs) show great promise because of their targeting action, lack of allergic reactions and serum sickness. The use of a cocktail of mAbs increases the "functional specificity" of their mixture, allowing them to bind to the active domains of different toxin chains and block their action. In this work, we obtained 14 murine mAbs to the catalytic and receptor-binding domain of botulinum toxin type A. The Sp2/0-Ag14 murine myeloma cell line and splenocytes from immunized mice of the BALB/c line were used as fusion partners. We have shown that the selected cocktail of three antibodies neutralizes native toxin more effectively than antibodies separately-complete neutralization is achieved at a toxin dose of 3LD50 and partial neutralization at 5LD50. We presume that this cocktail may be promising as a prototype for the creation of a therapeutic drug capable of neutralizing the toxin in the blood of patients.

Molecular Lipopolysaccharide Di-Vaccine Protects from Shiga-Toxin Producing Epidemic Strains of Escherichia coli O157:H7 and O104:H4.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O104:H4 strains are important causative agents of food-borne diseases such as hemorrhagic colitis and hemolytic-uremic syndrome, which is the leading cause of kidney failure and death in children under 5 years as well as in the elderly. METHODS: the native E. coli O157:H7 and O104:H4 lipopolysaccharides (LPS) were partially deacylated under alkaline conditions to obtain apyrogenic S-LPS with domination of tri-acylated lipid A species-Ac3-S-LPS. RESULTS: intraperitoneal immunization of BALB/c mice with Ac3-S-LPS antigens from E. coli O157:H7 and O104:H4 or combination thereof (di-vaccine) at single doses ranging from 25 to 250 µg induced high titers of serum O-specific IgG (mainly IgG1), protected animals against intraperitoneal challenge with lethal doses of homologous STEC strains (60-100% survival rate) and reduced the E. coli O157:H7 and O104:H4 intestinal colonization under an in vivo murine model (6-8-fold for monovalent Ac3-S-LPS and 10-fold for di-vaccine). CONCLUSIONS: Di-vaccine induced both systemic and intestinal anti-colonization immunity in mice simultaneously against two highly virulent human STEC strains. The possibility of creating a multivalent STEC vaccine based on safe Ac3-S-LPS seems to be especially promising due to a vast serotype diversity of pathogenic E. coli.

Characterization of the adaptive immune response of donors receiving live anthrax vaccine.

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors' blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

Immunological markers that correlate with protection immunity against tularemia infection.

An efficient immune response to tularemia is dependent on a strong cell-mediated component. We tried to identify markers of cellular immune responses that indicate a vaccine efficacy against tularemia. BALB/c mice were immunized with mutant F. tularensis 15∆23A and/or F. tularensis 15 NIIEG strains and then were challenged i.n. with F. tularensis Schu. We compared the influence of F. tularensis antigens (tularinum) in vitro on production of IL-1, IL-5, IL-6, IL-17, IFN-γ, and TNF-α by splenocytes obtained from intact mice and mice immunized with mutant F. tularensis 15∆23A and/or F. tularensis 15 NIIEG strains. We also compared expression of CD28, CD154, TLR-2, and CD69 markers on CD4 and CD8 T-cells after activation with tularinum in vitro. We found that tularinum-induced CD4(+) T-cells increased TNF-α and IFN-γ synthesis and expression of CD69 only in group mice with high degree of post immunization protection against F. tularensis Schu challenge. Estimation of CD69 expression on CD3(+)CD4(+) cells and IFN-γ, TNF-α synthesis by CD4(+) T-lymphocytes could be useful for determination protect ability of antitularemia immunity.