Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study.

Several candidate vaccines against Shigella spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered Shigella vaccine development over the past several decades. Bioconjugation technology, exploited here for an Shigella flexneri 2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel S. flexneri 2a bioconjugate vaccine made of the polysaccharide component of the S. flexneri 2a O-antigen, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 µg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant S. flexneri 2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against S. flexneri 2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to S. flexneri 2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.).

In vitro inflammation inhibition model based on semi-continuous toll-like receptor biosensing.

A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an in vivo-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., Pseudomonas aeruginosa) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-κB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests.

[A morphological evaluation of the protective properties of noninvasive recombinant strains of Shigella]. / Morfologicheskaia otsenka protektivnosti neinvazivnykh rekombinantnykh shtammov shigell.

In 2-3 weeks after the oral immunization of rabbits, made in one or two administrations, with attenuated two-marker S. dysenteriae 1 strain VS-12 and recombinant S. dysenteriae VS-12/S. sonnei NR-18 and S. flexneri y433/S. sonnei NR-18 pronounced immunological reaction developed in the mucous membrane of the small intestine: blast transformation follicles of Peyer's patches, an increase in the number of lymphoblasts and plasmocytes in the cupolae of follicles and in intestinal villi, and an increase in the number of lymphocytes and macrophages in the intestinal epithelium with their release into the lumen of the intestine after challenge with virulent shigellae. The protective potency of these recombinants after challenge with massive doses of virulent shigellae was found to be high, which was shown by quantitative evaluation of the decrease of adhesion, invasiveness and cytotoxicity, suppression of epithelial lesions and development of inflammation in the intestinal mucosa.

[The effect of the synergistic action of enterotoxin on the specific protective complex of Shigella sonnei]. / Effekt sinergentnogo deistviia énterotoksina na spetsificheskii protektivnyi kompleks Shigella sonnei.

The study has first established that enterotoxin enhances the protective potency of S. sonnei specific protective complex. This effect has been revealed both in experiments of the oral immunization of mice and in experiments of the conjunctival immunization of guinea pigs and depends on the dose of enterotoxin used in the experiment. The increase of protection has a specific character. These observations open prospects for further enhancement for the protective properties of S. sonnei specific protective complex, which should be taken into consideration in developing the vaccinal preparation.

[The action of ribosomal preparations on nonspecific resistance to bacterial infection and on early tolerance to endotoxic shock]. / Deistvie ribosomal'nykh preparatov na nespetsificheskuiu rezistentnost' k bakterial'noi infektsii i ranniuiu tolerantnost' k éndotoksicheskomu shoku.

Ribosomal preparations from Shigella flexneri and Shigella sonnei, introduced parenterally into mice, enhance their resistance to infection with the causative agents of typhoid fever and staphylococci. This effect is considerably less pronounced than that produced by the preparation of homologous lipopolysaccharide isolated by Boivin's method. After the administration of ribosomes nonspecific resistance to bacterial infective agents lasts for a short time. Ribosomal preparations do not enhance the resistance of mice to the lethal action of endotoxin.

[Specificity of the protective action of a ribosomal Shigella vaccine and the absence of activity in the ribosomes from R mutants]. / Spetsifichnost' protektivnogo deistviia ribosomal'noi shigelleznoi vaktsiny i otsutstvie aktivnosti u ribosom iz R-mutantov.

The ribosomal preparations of S. sonnei and some other bacterial species were obtained by the method of differential centrifugation, and the specificity of their protective action was studied in the keratoconjunctivitis test on guinea pigs. The ribosomal preparations were introduced parenterally in a single injection, and their protective action was determined two weeks later by the challenge of the animals with S. sonnei virulent strain and the subsequent calculation of the efficiency index (EI) by the formula: EI = C-V/C X 100, where C and V are the percentage of resistant eyes in the control and vaccinated groups of the animals respectively. For the ribosomal preparation obtained from a homologous avirulent strain this index was equal to 58%, while for the heterologous ribosomes obtained from Escherichia coli, Salmonella minnesota and S. flexneri in was close to zero. The ribosomal preparations obtained from S. sonnei R-strain which had no surface or cytoplasmic O-antigen also proved to be ineffective in rendering protection against local Shigella infection. The results of this investigation are compared with the data obtained by other authors, and the analysis of these results leads to the conclusion that the O-specific component is the indispensable factor of the protective activity of many ribosomal vaccines and its molecular properties require further study. The possible role of other components of the ribosomal vaccine is also discussed.

[Biological properties of a ribosomal vaccine from S. sonnei obtained by polyethylene glycol fractionation]. / Biologicheskie svoistva ribosomal'noi vaktsiny iz S. sonnei, poluchennoi metodom fraktsionirovaniia poliétilenglikolem.

Shigella ribosomal vaccine was prepared by fractionation with polyethylene glycol (PEG), recently proposed as an alternative of the more expensive and labor-consuming technique of differential centrifugation. In the present investigation the biological activity of ribosomal preparations isolated by these two methods was compared. Ribosomal vaccine obtained by PEG fractionation proved to be nontoxic for mice (LD50 greater than 2 mg) and produced no local and systemic reactions in monkeys when introduced subcutaneously in a dose of 600 micrograms. Ribosomes isolated by the two methods did not differ in the antigenic potency of their O-specific component responsible for inducing antibody formation in guinea pigs and monkeys. The protective potency of Shigella ribosomal vaccines prepared by PEG fractionation and ultracentrifugation was compared by tests in guinea pigs under the conditions of intraconjunctival challenge 2 weeks after a single injection of 40-200 micrograms of ribosomes. The mean resistance rate (percentage of protected eyes) was almost the same with both preparations, 67% and 65%, while in the control (nonimmunized) group only 13% of eyes were resistant to challenge. In monkey tests two injections of ribosomal vaccine obtained by PEG fractionation ensured a high protective effect against dysentery. No clinical signs of dysentery were observed in two groups of the test animals (totaling 10 monkeys) immunized 5 and 16 weeks before oral challenge with a dose of 75 X 10(9) virulent shigellae, which caused dysentery of moderate severity in all 5 control monkeys. The low toxicity and high protective potency of ribosomes isolated from S. sonnei by PEG fractionation makes it possible to use this method for the large-scale production of ribosomal vaccine.

Cytotoxicity of human peripheral blood and colostral leukocytes against Shigella species.

We examined the ability of human peripheral blood leukocytes to kill strains of Shigella sonnei and Shigella flexneri by using a modified bactericidal assay. Antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in the presence of specific rabbit immune serum directed against S. sonnei. With peripheral blood leukocytes from adults, ADCC was found only in the mononuclear cell and purified lymphocyte populations. Monocyte-macrophages and polymorphonuclear leukocytes were unable to demonstrate ADCC. Lymphocyte ADCC, which was not affected by the addition of phenylbutazone (an inhibitor of phagocytosis), was mediated by a non-T, Fc receptor-positive, HNK-1- cell. ADCC (using antiserum directed against virulent S. sonnei) was demonstrated against virulent S. sonnei but not against virulent S. sonnei or virulent S. flexneri. In contrast to leukocytes from adults, both mononuclear and polymorphonuclear cells from neonatal cord blood and from a patient with chronic granulomatous disease mediated anti-Shigella ADCC. Breast milk leukocytes (BMLs) collected 1 to 3 days postpartum were used as effector cells against virulent S. sonnei. The entire BML population, BMLs which did not adhere to plastic and BMLs which passed through nylon wool columns mediated both natural killer cytotoxicity and ADCC. In paired experiments, natural killer cytotoxicity and ADCC were significantly lower (30 to 45% inhibition) but not ablated, when phenylbutazone was added to BMLs and nylon wool-purified BMLs (P less than 0.05). These experiments suggest that colostral leukocytes mediated both extracellular and intracellular bacteriolysis in the presence and absence of specific antiserum. These mechanisms may be active in vivo in protection against shigellosis.

[Neutralizing action of human blood preparations on Shigella enterotoxins and choleragen]. / Neitralizuiushchee deistvie preparatov krovi v cheloveka na énterotoksiny Shigella i kholerogen.

The possibility of neutralizing the enterotoxic activity of S. dysenteriae 1 neurotoxin, S. sonnei live virulent cultures and cholerigen with immune sera of different animals, normal human sera and commercial gamma-globulin preparations is shown.

[Comparative immunomorphological study of the immunogenic activity of dysentery vaccines produced by different methods]. / Sravnitel'noe immunomorfologicheskoe izuchenie immunogennoi aktivnosti dizenteriinykh vaktsin razlichnogo sposoba izgotovleniia.

The morphological study of the ophthalmic mucosa of guinea pigs immunized locally with different dysentery vaccines has demonstrated the advantages of live dysentery vaccine prepared from Shigella sonnei 6S over heated vaccine and Shigella antigen extracts. The protective properties of dysentery vaccines, their capacity for protecting the mucous membrane from the penetration and intracellular multiplication of shigellae correlates with the degree of the manifestation of vaccine-induced plasmatocellular reaction in the epithelial and subepithelial zones. The importance of the virulence of the strains used for the preparation of vaccines, as well as the method of their preparation, for the immunogenic potency of vaccines is shown.

[Experimental study of the preventive properties of immune milk containing Shigella antibodies]. / Eksperimental'noe izuchenie preventivnykh svoistv immunnogo moloka, soderzhashchego protivoshigelleznye antitela.

The protective properties of the mammary gland secretions of cows immunized with Shigella sonnei into the udder were studied. As a model for this study the intranasal and intraperitoneal infection of white mice was used. Immune milk was found to have pronounced protective properties against S. sonnei. When introduced intraperitoneally, this milk protected the animals infected with S. sonnei from death. When introduced intranasally, it not only protected the animals from death, but perceptibly inhibited the development of the pathological process in the pulmonary tissue, preventing the multiplication of shigellae and accelerating the elimination of the infective agents from the lungs of the infected animals. As a rule, the degree of protective action was determined by the level of antibodies to shigellae in the substrate under test.

[Enteric dysentery vaccines and their effectiveness in a rabbit intestinal loop model. I. The residual virulence of attenuated vaccinal strains of Shigella administered into the intestinal loops]. / Izuchenie énteral'nykh dizenteriinykh vaktsin i ikh éffektivnosti na modeli kishechnykh petel' krolika. Soobshchenie I. Ostatochnaia virulentnost' attenuirovannykh vaktsinnykh shtammov shigell pri vvedenii v kishechnye petli.

Virulent Sh. flexneri strain 2a, Sh. sonnei strain, attenuated Sh. flexneri vaccine strain 2a 516M, and Sh. sonnei vaccine strain 6S (isolated by Yu. A. Belaya), as well as streptomycin-dependent Sh. flexneri strain 2a 1605/3 (isolated by V. V. Sergeev) were introduced into the ligated loops of the rabbit ileum. The use of light and immunofluorescent microscopy, the measurement of the volume of the fluid in the intestinal loops and the quantitative inoculation of their contents resulted in revealing the differences in the properties of the virulent and vaccine strains. The vaccine strains, in contrast to the virulent strains, did not proliferate in the lumen and did not cause the accumulation of fluid in the intestinal loops. They retained sharply limited, especially in the streptomycin-dependent bacteria, ability to penetrate into enterocytes and, via their cytoplasm, into the basement membrane, but lost their ability to proliferate in the cytoplasm of enterocytes (and probably even deteriorated there) and to cause plurulent ulcerous inflammation. This indicates that vaccine strains have insignificant residual virulence and suggests that the intestinal loop models, together with other models, may be used for testing the safety of vaccines prepared from Shigella strains.

[Activity of bovine colostral IgG in the human digestive tract]. / Aktivnost' v pishchevaritel'nom trakte cheloveka korov'ego molozivnogo IgG.

The biological activity of cow IgG administered orally to 11 volunteers in the colostrum of cows vaccinated with Sh. sonnei was studied. At the same time the degree of immunologic protection was determined on the experimental model of pepsin fragments of this IgG active against Sh. sonnei. IgG, introduced orally, could be regularly found in gastric and intestinal juices, as well as in coprofiltrates. A decrease in the concentration of IgG and related antibodies was observed as this IgG moved down along the digestive tract: the maximum loss of biological activity occurred in the lower sections of the intestine. The products of pepsin hydrolysis of immune cow IgG ensure pronounced protection against Sh. sonnei. The preservation of the biological activity of IgG in the digestive secretions of adults receiving immune colostrum orally indicates the expediency of further studies in the field of passive enteral immunization for the prevention and treatment of acute intestinal diseases.