Evaluation of vaccinal immunogenesis by the peroxidase method

The possibility of evaluating functional immunomorphogenesis in the course of the vaccinal process after the injection of conjugated and live brucellosis vaccines, as well as conjugated plague antigen and Yersinia pestis strain EV, to guinea pigs has been shown by means of the direct and two-layer variants of the immunoperoxidase assay. The dynamics of the accumulation of globulin-producing cells in the immunocompetent organs and the time course of immunoglobulin titers in the peripheral blood after the injection of live and conjugated vaccines have been followed up. These data may be used for the morphological evaluation of approved preparations.     

Comparative pathomorphologic study of the toxic properties of a corpuscular pertussis vaccine and of a cell-free pertussis preparation

The comparative study of morphological changes in the body of outbred mice under the action of corpuscular pertussis vaccine and acellular pertussis preparation has been made. The corpuscular vaccine has been shown to produce a pronounced, dynamically increasing toxic effect, thus causing the damage of lymphoid thymic and spleen cells, prolonged interstitial reaction in the lungs, destructive inflammatory process at the site of injection. The acellular pertussis preparation is less toxic, induces less pronounced changes in these organs at the early period of the experiment, stimulates the proliferation of lymphoid cells and lymphoblast transformation. As noted in this study, the damaging action of pertussis vaccine is mainly indicated by pathological phenomena appearing in the organs of the immune system, pulmonary parenchyma and muscular tissue (in the inoculation zone).     

Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain

We used the killing of Galleria mellonella (Lepidoptera: Pyralidae; the greater wax moth) caterpillar by the live vaccine strain (LVS) of Francisella tularensis to develop an invertebrate host system that can be used to study F. tularensis infection and the in vivo effects of antibacterial compounds on F. tularensis LVS. After injection into the insect hemocoel, F. tularensis LVS, killed caterpillars despite the association of LVS with hemocytes. The rate of killing depended on the number of bacteria injected. Antibiotic therapy with ciprofloxacin, levofloxacin or streptomycin administered before or after inoculation prolonged survival and decreased the tissue burden of F. tularensis in the hemocoel. Delayed drug treatment reduced the efficacy of antibacterials and especially streptomycin. The G. mellonella-F. tularensis LVS system may facilitate the in vivo study of F. tularensis, efficacy with antibacterial agents.     

Comparative study of the antigenic activity and allergic transformation of the body in human revaccination with different doses of a chemical brucellosis vaccine

The study of different doses of chemical brucellosis vaccine (0.5 mg, 0.75 mg and 1 mg), used for the booster immunization of persons who had received primary immunization with chemical and live brucellosis vaccines, revealed that the characteristics of its antigenic potency were somewhat higher after primary immunization with the live vaccine. When used for booster immunization, the tested doses showed no differences in their antigenic potency irrespective of prior immunizations received by the immunized persons. In booster immunization chemical brucellosis vaccine was found to have poorly pronounced allergenic properties irrespective of its booster dose with the tendency towards greater sensitization to brucellosis allergen in persons primarily immunized with the live vaccine. When considering the possibility of using brucellosis chemical vaccine in medical practice, the complex evaluation of the characteristics showing its reactogenicity and antigenic potency, as well as the allergic transformation of the body induced by the injection of the vaccine, was carried out, which made it possible to recommend 1 mg of the vaccine as the optimal booster dose.     

Deletion of IglH in virulent Francisella tularensis subsp. holarctica FSC200 strain results in attenuation and provides protection against the challenge with the parental strain

Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.     

Degree of chromatin activity and the RNA level in lymphocytes of popliteal lymph nodes of dogs after administration of an antigen]

In the experiment performed on 127 dogs by means of cytospectrofluorometric analysis, using fluorochrome acridine orange in dynamics up to 1 year, changes in the level of chromatin activation and RNA content have been studied in lymphocytes of the germinative centers and the crown of lymphoid nodules, in the paracortical zone and medullary cords of the regional and contralateral popliteal lymph nodes, after subcutaneous injection of antigen (BCG vaccine, 0.2 mg/kg) into the lateral area of the foot of the left pelvic extremity. The immune response is accompanied with a periodical increase in the level of chromatin activation and RNA content in populations of lymphocytes in the regional and contralateral popliteal lymph nodes with maximum in 6 h, 3-7 days, 1-3 months after the antigen injection. The intensity of these processes has an unequal level in lymphoid cells of various structural components; it is higher in lymphocytes of the contralateral lymph node.     

Virulence determinants and protective antigens of Francisella tularensis

Very little is known about virulence mechanisms of the highly virulent bacterium Francisella tularensis. Specific genetic features of F. tularensis have been obstacles for the development of effective tools for genetic manipulation. However, recent genomic sequencing and large-scale proteomic work have resulted in a substantial increase in the knowledge of F. tularensis. There is also a paucity of information on potential vaccine candidates. Recent work assessing the protective efficacy of the F. tularensis lipopolysaccharide has resulted in important contributions to the understanding of host-protective mechanisms. T-cell-mediated immunity appears to be crucial to protect against virulent F. tularensis strains. Few other vaccine candidates have been identified.     

Delayed type hypersensitivity reaction in the pathogenesis and formation of immunity in tularemia infections in mice

As a result of our studies, strain differences in the sensitivity of CBA and BALB/c mice to partially attenuated Francisella tularensis strain have been revealed. Relationship between the increased migration of lymphocytes to the liver and lymphoid organs and the intensive development of cell-mediated immunity reactions has been shown. An important role of local reactions (the skin at the site of the inoculation of F. tularessis + a regional lymph node) in the development of the pathological process and the formation of immunity to tularemia infection has been noted. A high level of resistance to F. tularensis strain used for inoculation in BALB/c mice seems to be greatly due to the fact that in these mice more intensive cell-mediated immunity reactions develop at the early stages of infection, than in CBA mice.     

Comparative study of the safety, reactogenicity and antigenic activity of chemical and live brucellosis vaccines in a controlled epidemiological trial

The complex evaluation of the reactogenicity characteristics revealed that after immunization with chemical brucellosis vaccine systemic reactions observed in most of the vaccinees were mildly pronounced and local reactions, mildly and moderately pronounced, their duration not exceeding 48-72 hours. During 4 months after immunization the antigenic and immunogenic potency of chemical brucellosis vaccine was no different from that of live brucellosis vaccine; seropositive persons immunized with chemical and live brucellosis vaccines showed no statistically significant differences in the geometric mean of their antibody titers, as determined in a number of serological tests, for a year after immunization. The examination of the vaccinees 4 and 12 months after immunization revealed that the sensitizing activity of chemical brucellosis vaccine was, respectively, 12.2 and 2.5 times lower than the allergenic action of live brucellosis vaccine.     

Recovery of the cellular immunity system after sublethal radiation

Dynamics and recovery of quantitative and qualitative characteristics of lymphoid tissue were studied after sublethal irradiation. The data obtained are indicative of a different dynamics of recovery of cells in central and peripheral lymphoid organs, GFUs and antibody-forming elements. The peculiarities have been revealed in a change of functional capacity of some T-lymphocyte populations.     

Effect of levamisole on the cellular and humoral immunity indices in patients with chronic brucellosis

In chronic brucellosis patients receiving levamisole and placebo the dynamics of quantitative and functional characteristics of cell-mediated and humoral immunity have been studied. The immunomodulating effect of levamisole, manifested only in the process of treatment by a rise in the number of circulating lymphoid cells, their functional capacity, a decrease in the disproportion of immunoregulatory cells and the amount of circulating immune complexes, has been established. The positive dynamics of immunological characteristics have been found to improve both immediate and remote results of the clinical effectiveness of levamisole.     

The characteristics of the antibody response of animals immunized with components of the surface structures of Francisella tularensis

Antibody formation in animals immunized with one of the components of F. tularensis surface structures was studied. The time course of antibody formation in 20 hamadryas baboons was studied in the passive hemagglutination (PHA) test, microagglutination (MA) test, and indirect enzyme immunoassay, used for the determination of IgG, IgA and IgM antibodies. The character of antibody response in the animals immunized with components of F. tularensis surface structures (S-complex) and with live tularemia vaccine was compared. The study revealed that immunization with the S-complex induced the formation of antibodies detected by all three methods. Antibody formation to the S-complex was found to be dose-dependent. With the increase of the injected dose of the S-complex, antibody titers determined in the PHA test decreased and those determined in the MA test increased, which was seemingly due to the induction of antibodies differing in their isotypes. After immunization with the S-complex the levels of IgG antibodies were lower and the levels of IgM antibodies by day 28 after immunization higher than after the injection of live tularemia vaccine.     

Cloning and expression of protective antigens of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Ag85B and ESAT-6 in <Emphasis Type="Italic">Francisella tularensis</Emphasis> 15/10

The possibility of expression of genes encoding mycobacterial antigens in Francisella tularensis 15/10 vaccine strain cells has been shown for the first time. To obtain stable and effective expression of mycobacterial antigens in the F. tularensis cells, the plasmid vector pPMC1 and hybrid genes consisting of the leader part FL of the F. tularensis membrane protein FopA and structural moieties of the mature protein Ag85B or the fused protein Ag85B-ESAT-6 were constructed. Recombinant strains F. tularensis RVp17 and RVp18 expressing protective mycobacterial antigens in the fused proteins FL-Ag85B and FL-Ag85B-ESAT-6, respectively, were obtained. Expression of the protective mycobacterial antigens in F. tularensis was analyzed using specific antisera to the recombinant proteins Ag85-(His)6 and ESAT-6-(His)6 isolated from Escherichia coli producer strains created on the basis of the pET23b(+) and pET24b(+) vectors. The expression of heterologous protective antigens in F. tularensis 15/10 is promising for creation of live recombinant anti-tuberculosis vaccines on the basis of the tularemia vaccine strain.     

Humoral factors of local and systemic immunity in the multiple peroral administration of a corpuscular pertussis vaccine

Multiple oral immunization with pertussis corpuscular vaccine was shown to lead to the considerable stimulation of local and systemic humoral immunity. The data on the titers of specific and normal secretory antibodies, on the levels of IgA in washings from the oral cavity, the small intestine and the lungs, on the titers of agglutinins and hemagglutinins in the blood serum, as well as on the morpho-functional transformation of the mucous membrane and the associated lymphoid tissue in the digestive tract, are presented in their dynamics. Specific pertussis antibodies in high titers were detected in both intestinal and pulmonary washings. The multiple administration of the vaccine did not produce pathological changes in internal organs.     

Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction

Proteome analysis of Gram-negative facultative intracellular pathogen Francisella tularensis (F. tularensis) live vaccine strain has been performed only on whole-cell extracts so far. This is the first study dealing with the analysis of the membrane subproteome of this microorganism. A fraction enriched in membrane proteins obtained by carbonate extraction was separated using two-dimensional electrophoresis and all visualized spots were identified by mass spectrometry. The reference map is the basis for further comparative analyses of virulent and non-virulent F. tularensis strains.     

Distribution and morphologic characteristics of lymphoid cells in the canine popliteal lymph nodes in their normal state and under antigen effect

By means of morphometry, light and electron microscopy methods peculiarities in distribution of small, middle and large lymphocytes, as well as plasmocytes in various zones of the popliteal lymph nodes have been studied in normal and in dynamics up to one year after subcutaneous injection of BCG vaccine into the left hind paw. The antigen produces certain changes in density and morphological parameters of lymphoid cells both in the regional and in the contralateral lymph nodes. For them 3 periods are specific. During the first 3 days they are not antigen-dependent (stipulated by the stress reaction), during 7-24 days antigen-dependent processes of proliferation and differentiation of lymphocytes get into action. In 3 months a new wave of the immune response is observed.     

The fate of intraperitoneally injected carbon particles in cyprinid fish

Summary The lymphoid organs of rosy barb (Barbus conchonius) and carp (Cyprinus carpio) were investigated for their phagocytic uptake of carbon, after its intraperitoneal injection. Carbon handling was similar in both species. It was first detected in the lymphoid organs at 30 min after injection. During the first day, carbon was phagocytized by macrophages situated in the spleen within the ellipsoids and in the red pulp. In head and trunk kidney, carbon was found in macrophages scattered throughout the haemopoietic parenchyma, and in cells lining the blood sinuses. In the spleen, macrophages replete with carbon left the ellipsoidal structures and formed aggregates with pigment-containing macrophages from day 6 onwards. In all lymphoid organs, almost all carbon was ultimately concentrated in the melano-macrophage centres.     

Evaluation of immunobiological activity of Francisella tularensis C-complex preparations as promising component of subunit vaccines

Data on influence of Francisella tularensis C-complex preparations on formation of immunity against tularemia are presented. Study of cellular immunity characteristics as well as dynamics of antibody response was carried out on white mice and guinea pigs models. Absence of toxicity, pyrogenicity, and negative effects on immunocompetent cells in combination with protective activity points to possibility of use the C-complex as a component of a subunit vaccine.     

Francisella tularensis vaccines

Francisella tularensis is the causative agent of tularaemia, a disease which occurs naturally in some countries in the northern hemisphere. Recently, there has been a high level of interest in devising vaccines against the bacterium because of the potential for it to be used as a bioterrorism agent. Previous human volunteer studies have shown that a strain of F. tularensis [the live vaccine strain (LVS)] that has been attenuated by laboratory passage is effective in humans as a vaccine against airborne disease. However, for a variety of reasons it seems unlikely that the LVS strain will be licensed for use in humans. Against this background there is an effort to devise a licensable vaccine against tularaemia. The prospects for a killed whole-cell subunit of live attenuated vaccine are reviewed. A rationally attenuated mutant seems the most likely route to a new tularaemia vaccine.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.