Level of IgG antibodies to bacteria of 12 species in the blood sera of rabbits, immunized with preparations obtained from Neisseria meningitidis, grown under the stress conditions

The blood sera of rabbits, immunized with preparations obtained from N. meningitidis of serogroups A, B or C, cultivated under the stress conditions, were studied. These sera were found to contain IgG antibodies not only to N. meningitidis antigens, but also to the bacterial antigens of 12 species. The sera of rabbits, immunized with meningococcal preparation of serogroup A, were found to have the elevated levels of IgG antibodies, in comparison with the control, to the antigens of 3 other bacterial species; the blood sera of rabbits, immunized with meningococcal preparation of serogroup B, were found the elevated levels of IgG antibodies to the antigens of 11 other bacterial species; and the blood sera of rabbits, immunized with meningococcal preparation of serogroup C, to the antigens of 9 other bacterial species. The study of serogroup B meningococci, used as an example, revealed the influence of the growth phase of the culture on the content of cross-reacting antigens. Their greatest amount was determined at the stationary phase when the stressor effect on the culture reached its maximum and their least amount, at the exponential phase when the stressor effect on the culture was minimal. It was, therefore, found to be expedient to obtain immunodiagnostic and test systems from N. meningitidis cultures, grown to middle of the exponential phase of growth.     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

Acellular pertussis vaccine

Protective, immunogenic, toxic, and sensitizing properties of acellular pertussis vaccine (aPV) developed according to original technology were studied, aPV had marked protective activity which lasted more than 2 years. Sera of mice immunized by aPV also possess protective properties, and they were more prominent than in sera of mice immunized by pertussis bacteria suspension (PS). Immune sera to aPV neutralized cytopathogenic effect of pertussis toxin (PT) on ovarian Chinese hamster cells in 1:250 dilution, whereas neutralizing activity of sera to PS was very low. Level of antibodies to PT was higher in rabbits immunized, according to schedules and dosage recommended for children, by aPV than by PS. High immunogenicity of aPV was proved also by levels of IgG to PT in sera of mice immunized three times by aPV in human dosage. During experiments on mice and guinea pigs aPV had mild toxicity, did not induce autoimmune process, did not have anaphylactogenic properties compared with bacterial suspension characterized by high anaphylactogenic activity. Histamine-sensitizing abilityof aPVwas 40 times lower than that of PS. Assessment of pyrogenic properties of aPV and PS performed on rabbits showed that aPV was 1,000 times less pyrogenic than PS. Obtained results demonstrate high protective and immunogenic properties of domestic acellular pertussis vaccine and its low toxic and sensitizing characteristics.     

Complement requirement of neutralizing antibodies in different classes of immunoglobulin appearing in rabbits and guinea pigs after primary and booster immunizations with herpes simplex virus.

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgS (7 S gamma2). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-merceptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

Appearance of Mouse-Lethal Toxin in Liquid Cultures of Bordetella pertussis

The mouse-lethal toxin present in liquid cultures of most smooth strains of Bordetella pertussis is known to originate in the cytoplasm of the organism but to be most lethal for mice when released into the supernatant fluid. It is also recognized that cell degeneration and lysis occur in liquid cultures during the stationary and decline phases of growth. For these reasons, it is generally believed that most of the toxicity demonstrable in liquid cultures at the time of harvest is released during the later stages of cultivation, when high alkalinity and aging of cells favor lysis. However, the results reported here have indicated that high levels of mouse-lethal toxicity arise during very early log phase and that the peak of toxicity is reached before the end of the log phase. No further increase in toxicity was observed during stationary and decline phases. The very early appearance of toxicity could not be explained by the presence in the inoculum of a proportion of dead and degenerating cells, and it is concluded that the toxin is produced mainly by actively growing cells. This was confirmed by tests on organisms growing in continuous culture. Electron-microscopic examination of cells from a very early log-phase culture revealed the presence of large numbers of small vesicles on the cell walls of about 5% of the population. It is suggested that these vesicles may be associated with the releases of toxin from living cells. It is concluded that no useful reduction in the toxicity of cultures would result from harvesting before the end of the log phase of growth.     

Complement Requirement of Neutralizing Antibodies in Different Classes of Immunoglobulin Appearing in Rabbits and Guinea Pigs after Primary and Booster Immunizations with Herpes Simplex Virus

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgG (7 S γ₂). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-mercaptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

Identification and the role of soluble antigens detected in bile from Eimeria stiedai-infected rabbits

Antibodies against Eimeria stiedai sporozoites and merozoites were detected in the sera of rabbits immunized with bile obtained from infected rabbits on the 15th day post-infection. The trails made by gliding sporozoites were also detected by the sera. After penetration into the host cell, an antibody-binding region was observed on the parasitophorous vacuole membranes of the parasites. Rabbits administered a combination of the bile and cholera toxin shed fewer oocysts in the feces after infection than control rabbits. The immunized rabbits developed a high level of IgA antibody against soluble antigens in the bile. By immunoblotting, antigens with molecular masses of 32, 37, and 49 kDa were detected in the bile obtained from infected rabbits on the 15th day postinfection. Absorption treatment with sporozoites reduced or abolished the antibody reactivity to the 32-kDa antigen of merozoites and the bile antigens. However, antibody reactivity to the 37- and 49-kDa antigens still remained. These results indicate that soluble antigens are present in the bile of rabbits in the acute phase of infection, and these may be produced and released by merozoites during the host cell invasion process.     

Humoral response to the injection of acellular Pertussis vaccine

The humoral response of mice and rabbits to the injection of whole-cell pertussis vaccine (PV) and acellular pertussis vaccine (APV), developed at the Mechnikov Research Institute for Vaccines and Sera (Russian Acad. Med. Sci.) in Moscow, was studied. In the sera of immunized animals antibodies to the antigenic complex were determined in the direct hemagglutination (DHA) test, specific antibodies to filamentous hemagglutinin (FHA) and pertussis toxin (PT)--in the enzyme immunoassay (EIA) and antibodies neutralizing PT in a cytopathogenic dose (CPD)--in neutralization test on Chinese hamster ovary (CHO) cells. In mice and rabbits immunized with APV the antibody titers determined in the DHA test were higher than those in the animals immunized with PV. Specific antibodies titers to FHA and PT in the sera of rabbits immunized with APV were also higher than those in the sera of rabbits immunized with PV. High dilutions of sera taken from the animals immunized with APC neutralized 4-16 doses of PT in the neutralization test on CHO cells. The most important result of this study was the detection of a more pronounced immune response in the animals immunized with APV in comparison with that induced by PV according to the results obtained in EIA and in the test of PT CPD neutralization on CHO cells.     

Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes

DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.     

Perspectives for development of polyvalent meningococcal vaccine

Experimental whole-culture oral polyvalent meningococcal vaccines against serogroups A, B and C consisting of three monovalent components in different proportions have been developed and evaluated. Kinetics of IgG response to meningococcal antigens (outer membrane proteins, polysaccharide, lypooligosaccharide of these serogroups) in sera of rabbits immunized orally with these preparations was measured. Sharp rise of IgG levels (on 2 - 3 orders) compared to baseline has been detected as well as persistence of high titers during the observation period (322 days).     

Induction of humoral immunity in response to immunization with recombinant Mycobacterium bovis BCG expressing the S1 subunit of Bordetella pertussis toxin

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.     

Antitoxin in human pertussis immune globulins

The level of antitoxin i.e. neutralizing antibodies to pertussis toxin, or lymphocytosis promoting factor, was determined in six pertussis immune globulin preparations from different manufactures. A comparison with antitoxin levels after natural pertussis disease in adults showed that pertussis immune globulins did not contain more antitoxin than convalescent phase sera, i.e. they had very low antitoxin content for specific immune globulins. Agglutinin and anti-FHA titres were relatively higher in immune globulins, probably reflecting a difference between the antibody response elicited by whole cell vaccines used for hyperimmunization in immune globulin production and by natural disease. The low antitoxin content of currently available pertussis immune globulin preparations could explain the inefficacy or conflicting results obtained with these products in prophylaxis and therapy of whooping cough.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. I. Antibody level for B pertussis antigens in children without respiratory tract infection symptoms

The aim of the first part of this study was to determine antibody level to pertussis toxin, filamentous hemagglutinin and endotoxin of B. pertussis in children without symptoms of respiratory tract infection. The serum samples obtained from 276 children (age range: 6 weeks-16 years) were examined using indirect hemagglutination and ELISA tests. Normal antibody levels to 3 B. pertussis antigens were determined for 95% of the serum samples as the upper cut-off levels depending on children age. Very high level of IgG antibodies to B.perussis antigens was observed in the control population. The lowest antibody level was found in IgA class to pertussis toxin and lipopolysaccharide. It was also established that the IgM level to 3 B. pertussis antigens was rising together with children age.     

Detection of noncapsular antigens in pneumococcus

Complex antigenic preparations obtained from noncapsular pneumococcal strains were used for the immunization of rabbits and guinea pigs. The injection of the preparations in complete Freund's adjuvant for 5 weeks led to the appearance of antibodies in their sera. The antibodies were detected by the double immunodiffusion test. The preparations obtained from different strains by extraction (with Triton X-100 or sodium deoxycholate) or by disintegration contain common pneumococcal antigens.     

Comparative study of the immunoglobulins and specific antibodies in the blood of rabbits immunized with ADTP vaccine and ADT-anatoxin with normal and reduced antigen contents

Adsorbed DPT vaccine and adsorbed DT toxoids with normal and reduced antigen content were used for the immunization of rabbits. The levels of IgM and IgG and the dynamics of antibodies to diphtheria and tetanus toxins and to Bordetella pertussis in the blood sera of the animals were studied in the postvaccinal period (on days 15 and 34). This study revealed that the reduction of the antigen content of adsorbed DT toxoid to 5 Lf of diphtheria toxoid and 5 binding units of tetanus toxoid did not decrease the capacity of the preparation for increasing the levels of IgG and IgM, antibodies to diphtheria and tetanus toxins in the sera of the rabbits. The reduced content of these toxoids in adsorbed DPT vaccine did not affect its capacity for inducing the enhanced synthesis of IgG, antibodies to diphtheria and tetanus toxins, while the production of IgM and IgA remained unchanged. At the same time an increase in the titers of antibodies to B. pertussis in the animals was less pronounced than that observed after the injection of commercial adsorbed DPT vaccine. Additional investigations are necessary in order to establish the protective potency of the pertussis component in adsorbed DPT vaccine with the reduced content of toxoids and to find out the optimum antigenic composition for this preparation.     

Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muramyl peptide

Synthetic antigens employed in experimental synthetic vaccines are generally small haptenic peptides. Therefore, effective immunization with these antigens usually requires the use of an immunogenic carrier. Tetanus toxoid has been proposed for use as a carrier in future synthetic vaccines due to its high immunogenicity and acceptance for human use. Previous studies employing standard hapten/carrier systems such as DNP/KLH have demonstrated, however, that an epitope-specific suppression occurs when mice previously primed with carrier are subsequently immunized with an haptenic epitope conjugated to the same carrier. These same studies have shown that Bordetella pertussis vaccine administered at the time of carrier priming abrogates epitopic suppression. In the present investigation, epitopic suppression was studied in a synthetic vaccine model employing tetanus toxoid as a carrier. Results from these studies indicated that mice primed with tetanus toxoid 1 month before immunization with a peptide-tetanus toxoid conjugate exhibited enhanced secondary anti-tetanus toxin responses but decreased anti-peptide responses. Furthermore, injection of pertussis vaccine or purified B. pertussis toxin or endotoxin at the time of carrier priming could block the establishment of epitopic suppression. Administration of B. pertussis components enhanced antibody responses to both the carrier and the synthetic peptides as compared with responses of control animals. In addition, administration of an adjuvant-active nonpyrogenic derivative of muramyl dipeptide. Murabutide, with carrier priming reduced epitopic suppression of anti-peptide responses. B. pertussis toxin or endotoxin administered to mice previously suppressed by carrier priming with the first injection of carrier-peptide conjugate overcame epitopic suppression with resultant titers of anti-peptide antibody equal to or greater than nonsuppressed controls. These results suggest that the use of adjuvants with future synthetic vaccines may contribute the additional advantage of overcoming epitopic suppression, thus permitting the use of common, well-tolerated carrier systems such as tetanus toxoid in synthetic vaccine preparations.     

Protective effect of antiganglioside antibodies against experimental Trypanosoma brucei infection in mice

Liposome-associated ganglioside antigens (ganglioside GM1 or bovine brain gangliosides) were prepared to facilitate the potential protective efficacy for Trypanosoma brucei. Mice were immunized with liposome-associated ganglioside GM1 or bovine brain gangliosides intraperitoneally (i.p.). After immunization, significantly higher antigen-specific IgG and IgM antibodies were detected in sera than in the nonimmunized control group. When sera from immunized mice were analyzed for isotype distribution, antigen-specific IgG1, IgG2a, and IgG3 antibody responses were also noted. After immunization, mice were challenged i.p. with 1 x 10(2) cells of T. brucei. Sixty percentage of liposome-associated ganglioside GM1-immunized mice survived the infection, and all the mice immunized with bovine brain gangliosides-containing liposomes survived. However, all control mice died within 7 days after infection. These data demonstrate that liposomes containing ganglioside antigens have the potential usefulness for the induction of a protective immune response against T. brucei infection and suggest the possibility of developing vaccines that may ultimately be used for the prevention of trypanosomiasis.     

The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.     

The properties of cell wall hydrolysates of Streptococcus group A]

Products obtained from lysis in the cell wall of group A streptococcus have been studied in different growth phases: at the end of the exponential phase and in the stationary one. Endo-beta-N-acetylmuramidase extracted from the culture liquid of Streptomyces levoris 96 has been used for lysis of streptococcus. It is stated that streptococcus cell walls isolated at different growth stages differ in the protein and polysaccharide content. High content of protein in the cell wall of a young culture makes lower the initial rate of the walls' hydrolysis by endo-beta-N-acetylmuramidase. However, with the enzyme penetration into peptidoglycan the rate of hydrolysis of cell walls gets higher and after four-hour incubation the lysis degree of walls of the 16- and 8-hour cultures reaches the equal value (63%). Studies in the protein composition of lysates of the streptococcus cell walls have shown that they contain at least 12 proteins most of which are acid and neutral ones.