Experimental autoimmune myasthenia gravis: can pretreatment with 125I-labeled receptor prevent functional damage at the neuromuscular junction?

Rats were immunized with purified receptor from electric fish to induce experimental autoimmune myasthenia gravis (EAMG). It is implied by the clonal selection theory that antigens react only with receptors on specific immunocompetent cell subpopulations. In an attempt to damage these specific cells with the aid of highly radioactive antigen, one group of rats was pretreated with an additional injection of radiolabeled receptor of high specific activity 3 days before the basic immunization. The success of the immunization was monitored by measuring changes in the following three parameters: antibody titers against nicotinic acetylcholine receptor; number of alpha-bungarotoxin-binding sites at endplates; and number of acetylcholine-operated ionic endplate channels, using quantitative electrophysiologic methods. Conventionally immunized animals showed the classical signs of EAMG: elevated antibody titers against nicotinic acetylcholine receptor and a reduction of the number of alpha-bungarotoxin-binding sites, as well as reduction of the number of acetylcholine-operated ionic channels. The same symptoms were found in animals pretreated with unlabeled receptor and in animals pretreated with radioactive albumin. Animals pretreated with radioactively labeled receptor showed far less reduction of functional nicotinic acetylcholine receptor and only slightly raised antibody titers. This study suggests that preimmunization with radioactive antigen selectively eliminates immunocompetent cells, thus precluding the production of antibodies by a subsequent immunization procedure. The same protective effect cannot be obtained by either preimmunization with unlabeled antigen or by radioactively labeled unspecific antigen.     

Elimination of Klebsiella pneumoniae and humoral immunity in immunization with the IS-4 immunostimulating mixture

The present investigation has revealed that in mice, immunized with preparation HC-4 (an immunostimulating agent consisting of water-soluble antigenic complexes obtained from 4 opportunistic microorganisms: Klebsiella pneumoniae, staphylococcus, Proteus and Escherichia coli K-100 having a common antigen with Haemophilus influenzae) and challenged with K. pneumoniae culture on day 7 after immunization, the complete elimination of K. pneumoniae from the blood occurs within 24 hours. The subcutaneous immunization of rabbits with the above preparation leads to a significant increase in antibody titers, determined in the passive hemagglutination test with Klebsiella diagnosticum. The test of the passive protection of mice from Klebsiella sepsis has revealed a rise in the preventive activity of the sera of rabbits immunized with this preparation.     

Specific cross-protective antigonococcal immunity in the murine genital tract

Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.     

Experimental study of the immunological activity of a preparation made from the antigenic complexes of opportunistic microorganisms administered by different routes

The data obtained in the experimental study of the humoral factors of local and systemic immunity, as well as the morphofunctional changes of internal organs, after multiple subcutaneous and aerosol immunization with the combined preparation of Proteus, Staphylococcus, Klebsiella pneumoniae and Escherichia coli antigens (preparation nC-4) are presented. The subcutaneous and aerosol administration of preparation nC-4 has been found to induce an increase in the levels of antibodies to all components of the preparation in the blood serum and in respiratory tract secretions. The introduction of the preparation through the respiratory tract resulted in an earlier intensive accumulation of specific antibodies and IgA in respiratory tract secretions. The results of the quantitative cytological study of respiratory tract secretions and the morphofunctional changes of the lymphoid tissue associated with the lungs are indicative of an important role played by cell-mediated immunity factors in the formation of local postvaccinal immunity.     

Macrophage transformation of mononuclear cells following primary and secondary experimental immunization with measles vaccine

Cell-mediated and humoral immune response was studied in guinea pigs receiving two immunizations with live measles vaccine l-16 in doses of 1000 TCD50/0.5 ml at an interval of 45 days. The results of this study showed that the maximum level of the macrophagal transformation of mononuclears and the most intensive synthesis of antimeasles antibodies were observed on day 10 after booster immunization. The intensification of cell-mediated and humoral immune response was found to depend on the initial immunological background. The animals having had high values of cell-mediated response before booster immunization showed a decrease in these values, while an increase in antibody titers in such animals was transitory.     

嗜麦芽寡养单胞菌脂多糖对斑点叉尾免疫保护作用

嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)是近年来引起斑点叉尾高致死性、传染性疾病的主要病原之一。为了研究嗜麦芽寡养单胞菌脂多糖对斑点叉尾的免疫保护作用,实验选用了400尾健康斑点叉尾,随机分成Ⅰ、Ⅱ、Ⅲ、Ⅳ四个组,每组用鱼100尾,每组下设两个重复,每个重复用鱼50尾,以腹腔注射的方式分别向Ⅰ、Ⅱ、Ⅲ、Ⅳ四个组的斑点叉尾注射嗜麦芽寡养单胞菌脂多糖、无花果多糖加脂多糖、全菌灭活苗和生理盐水,在试验第0、第28天时分别进行首次免疫和加强免疫,试验期间每隔7d,对试验鱼的血液白细胞杀菌活性、补体C3含量、IgM含量和血清凝集效价滴度进行测定;试验第49天时进行活菌攻毒。结果表明,首免后,接种脂多糖、无花果多糖加脂多糖的试验鱼血清凝集效价滴度明显升高,白细胞杀菌活性、补体C3含量、IgM含量也明显增加;加强免疫后,脂多糖、无花果多糖加脂多糖的试验鱼血清凝集效价滴度峰值分别为1∶256和1∶512,白细胞杀菌活性峰值分别为0.565和0.511,补体C3含量峰值分别0.194和0.180mg/mL,IgM含量峰值分别1.415和1.464mg/mL,免疫保护率分别为70.0%和65%。嗜麦芽寡养单胞菌脂多糖和脂多糖+无花果多糖受免鱼的上述指标均显著(P<0.05)或极显著(P<0.01)地高于生理盐水注射组,这两者的免疫保护效果优越于全细胞灭菌苗。     

Duration of immunity in children who received a primary complex of inoculations with ADT-M anatoxin and an assessment of postponed secondary revaccination against diphtheria and tetanus (the results of an epidemiologic trial)

The prolonged observations of the immunological effectiveness of adsorbed diphtheria-tetanus toxoid with reduced antigen content in children who had received the primary course of immunization with this preparation showed that the preparation induced the development of prolonged and intensive immunity to both infections. In 2-3 years after the first booster immunization the protective level of diphtheria antitoxin was registered in 89.9% and that of tetanus antitoxin, in 99% of children. 6 years later the level of immunity remained practically unchanged: the titers of diphtheria antitoxin above the protection level were determined in 92% and those of tetanus antitoxin, in 97% of children. These data made it possible to increase intervals between booster immunizations to 6-7 years in children of this category. The results of the epidemiological trial made to find out the possibility of a change in the timing of the second booster immunization confirmed the expediency of postponing booster immunization from 6 and 11 years to 9 and 16 years of age.     

Immunoprevention of staphylococcal infections at an industrial enterprise

The immunogenicity, immunological and epidemiological effectiveness of the subcutaneous, enteral and combined immunizations of the workers at a steel plant with staphylococcal toxoid has been studied. The subcutaneous injection of the adsorbed toxoid in a dose of 0.2 ml by means of the jet injector, model, was accompanied by moderate reactogenicity (the frequency of local reactions was 26.8%, and the frequency of systemic reaction 18%) and produced 4-fold increase in antitoxin titers and 12-fold decrease in the morbidity rate of staphyloccal infection. Enteral immunization in 3 administrations proved to be nonreactogenic and only slightly effective (antitoxin titers increased more than 2-fold, morbidity rate fell 2-fold). Combined immunization (subcutaneous and enteral) with the toxoid, preceded by the injection of gamma globulin, reduced the frequency of reaction 2- to 3-fold and stimulated antitoxin production (the titer increased 8-fold), thus decreasing the morbidity rate of staphylococcal infection 3.1-fold.     

Production of antibodies to sheep red blood cells and Brucella abortus in Mongolian gerbils (Meriones unguiculatus).

The levels of total and 2-mercaptoethanol (2-ME)-resistant antibodies in male Mongolian gerbils (Meriones unguiculatus) to sheep red blood cells (SRBC) were higher than those in male C57BL/6 mice after the first and second immunizations. On the other hand, the total antibody level to Brucella abortus (BA) in gerbils was comparable to that in mice, whereas 2-ME-resistant antibody titers were lower after the first and second immunizations than in mice. After injection of 8 x 10(4) SRBC, male gerbils did not produce either total or 2-ME-resistant antibodies after the first immunization, but they produced total and 2-ME-resistant antibodies after the first to the fourth immunizations with different dilutions of BA, and both types of antibody titers significantly increased with the repeated immunizations. There were no sex differences in total and 2-ME-resistant antibody production to SRBC.     

Immunization of sheep against infection with larvae of the blowfly Lucilia cuprina

Sheep were repeatedly exposed to a second stage excretory-secretory antigen preparation of Lucilia cuprina by intradermal injection (S group) or intranasal aerosol (I group) in an attempt to induce immunity to the larvae. Hypersensitivity responses to the injections were monitored and correlated with larval numbers at subsequent challenge. Intradermal injections showed that the immediate or IgE-mediated and the intermediate or Arthus response were the major skin hypersensitivity reactions to the larval antigens. At challenge there was no significant reduction in larval numbers between the S and the control group, however the Arthus reaction did show some correlation with larval recoveries in the S group. There was a significant reduction in larval numbers (P < 0.005 Mann-Whitney U Test) between the I and the control group. In addition, the animals which showed respiratory hypersensitivity to the aerosol during the immunization period had the lowest larval recoveries. The results of a second challenge in the I group did not show continued protection. It is suggested that the protective response was suppressed by exposure to antigens at the first challenge infection. Exudate samples recovered during the infection were analysed for protein amount and the results were correlated with larval survival. They suggested that two separate mechanisms of resistance are operating in this experiment. The first occurs early in the infection, is probably associated with immediate hypersensitivity, and may control the initiation of protein leakage. The second occurs later in the infection and may result in the leakage of proteins able to control larval survival.     

Immunization of rabbits with recombinant Treponema pallidum surface antigen 4D alters the course of experimental syphilis

Rabbits immunized with the protease resistant recombinant Treponema pallidum surface-associated Ag 4D showed an altered course of experimental syphilis after intradermal challenge with virulent T. pallidum. Vaccination trials using three different protocols were examined. In one experiment, a combined i.m.-i.v. immunization protocol was compared with an i.m. injection schedule. Four of five rabbits immunized by the i.m.-i.v. protocol developed morphologically atypical lesions at each of 16 sites, whereas the i.m. injected animals showed no evidence of attenuated disease. Moreover, immunization by both protocols elicited equally high ELISA titers of 4D-specific IgG antibody suggesting that cellular immune mechanisms may have been involved. In an effort to augment cell-mediated responses to 4D, seven rabbits received i.v. immunizations with an emulsion of 4D, purified BCG cell wall skeletons, and trehalose dimycolate in an oil-microdroplet form (4D-cell wall skeleton-trehalose dimycolate). Splenic lymphocytes from three representative immunized animals had a strong in vitro proliferative response to 4D as compared to controls. Further, all animals developed high anti-4D titers in response to immunization. After challenge with virulent T. pallidum, six of the seven rabbits developed an early cutaneous response (3-8 days) at the sites of inoculation consistent with a hypersensitivity reaction followed by morphologically atypical lesions. Aspirates from each of three representative atypical lesions were devoid of treponemes by darkfield examination at day 14, whereas motile T. pallidum were observed in aspirates from four of five control sites. Between days 20 and 24, lesions in the 4D-cell wall skeleton-trehalose dimycolate immunized rabbits began to enlarge and become more typical in appearance; aspirates from six of seven representative lesions were darkfield positive. We conclude that 1) immunization with the r4D Ag alters the course of experimental syphilis in a manner consistent with previously defined parameters of partial protection and 2) immunizations protocols containing an i.v. component are more effective than protocols using the i.m. route alone.     

Immunization with outer-membrane protein(s) of Bacteroides fragilis: an experimental study in mice

A crude outer-membrane protein (OMP) preparation from a strain of Bacteroides fragilis, grown in supplemented brain-heart infusion broth, was tested for its protective effect against subcutaneous infection in mice. Immunization with six doses, each of 100, 150 or 200 g OMP preparation, gave some protection: abscesses completely disappeared 15 to 22 days after immunization. In non-immunized animals and animals immunized with doses of 10, 20, 40 or 80 g each, well demarcated abscesses were seen beyond day 22 post-immunization. Although crude OMP elicited good antibody response, with maximum titres on day 4 post-immunization, high titres could not be associated with healing of the abscesses.     

Evaluation of a subcutaneously implanted chamber for antibody production in rabbits.

Polyclonal antibody production in subcutaneous chambers was compared to traditional antibody production methods in rabbits. The chamber, a sterilized plastic wiffle golf ball that had been surgically implanted in the subcutis of the thoracic region, was immunized via a percutaneous injection of antigen into the core of the ball through one of the perforations in the chamber wall. Rabbits bearing chambers were immunized on the same schedule and with the same concentrations of antigens as were provided the adjuvant injected rabbits. Fluid volumes of 12 to 22 ml could be removed from each chamber at weekly intervals. Chamber antibody to specific microbial antigens was equal to or better than serum antibody produced to the same antigens with Freund's or acrylamide adjuvants. The comfort of the rabbit, the ease in chamber immunization, and the recovery of high titer antibody in large volumes make the subcutaneous chamber an attractive method for polyclonal antibody production.     

Sensitizing and protective activity of a staphylococcal vaccine made of water-soluble antigens, depending on the method and schedule of administration

An experiment on guinea pigs immunized with staphylococcal vaccine prepared from water-soluble antigens revealed that the degree of developing sensitization and specific resistance was essentially determined by the method and schedule of the administration of the preparation. The intranasal administration of the vaccine induced a lesser degree of sensitization in comparison with its subcutaneous injection. The optimum response to the administration of the vaccine (a low sensitization level and a high degree of protection from infection) was observed in the animals immunized first intranasally and then by subcutaneous injection. The subcutaneous injection of the preparation in combination with its subsequent intranasal application induced a more pronounced degree of sensitization and a lesser degree of protection from infection.     

Assessment of the effectiveness of different methoods of immunization with live plague vaccine EB in aerosol infections]

The work deals with the results of the comparative evaluation of the effectiveness of vaccines developed at the Sanitary Research Institute (Zagorsk) and the Mechnikov Research Institute for Vaccines and Sera (Moscow), as well as two methods of immunization against plague, by inhalation and subcutaneous injection, under the conditions of aerosol infection. The immunogenic effectiveness of both vaccines, when evaluated in terms of LD50, was shown to be approximately the same, but the animals immunized by the inhalation method with the vaccine developed at the Sanitary Research Institute proved to be less susceptible to infection than those immunized with the vaccine developed at the Mechnikov Research Institute for Vaccines and Sera in Moscow. After immunization by the inhalation method the vaccine developed at the Sanitary Research Institute rendered more effective protection (3- to 4-fold) against aerosol infection than after immunization by subcutaneous injection. The animals immunized by the inhalation method proved to be capable of surviving plague in the primary pneumonic form.     

The evaluation of the reactogenicity and immunogenic activity of a new concentrated inactivated leptospirosis vaccine

In the controlled field trial the reactogenicity, safety and antigenic activity of a new concentrated inactivated leptospirosis vaccine after its administration in one and two injections of 0.5 ml were studied in comparison with those of the existing commercial vaccine, introduced in two injections in doses of 2.0 and 2.5 ml. The new experimental vaccine exhibited low reactogenicity and was found to be safe and highly immunogenic when introduced in a single injection of 0.5 ml. As shown in this trial, the immunogenic characteristics of immunization made in a single injection were not inferior than those obtained as the result of immunization made in two injections, yielding high percentage of seroconversions (89.8% to 98.3%) with respect to 4 Leptospira serogroups and leading to the production of the protective titers of corresponding antibodies. The existing commercial vaccine was inferior to the experimental one in antigenic activity (the frequency of seroconversions, antibody titers). The results of the trial make it possible to recommend the experimental concentrated leptospirosis vaccine for use in medical practice in a dose of 0.5 ml introduced in a single injection.     

Extended immunization intervals enhance the immunogenicity and protective efficacy of plasmid DNA vaccines

Effective vaccines against infectious diseases and biological warfare agents remain an urgent public health priority. Studies have characterized the differentiation of effector and memory T cells and identified a subset of T cells capable of conferring enhanced protective immunity against pathogen challenge. We hypothesized that the kinetics of T cell differentiation influences the immunogenicity and protective efficacy of plasmid DNA vaccines, and tested this hypothesis in the Plasmodium yoelii murine model of malaria. We found that increasing the interval between immunizations significantly enhanced the frequency and magnitude of CD8+ and CD4+ T cell responses as well as protective immunity against sporozoite challenge. Moreover, the interval between immunizations was more important than the total number of immunizations. Immunization interval had a significantly greater impact on T cell responses and protective immunity than on antibody responses. With prolonged immunization intervals, T cell responses induced by homologous DNA only regimens achieved levels similar to those induced by heterologous DNA prime/ virus boost immunization at standard intervals. Our studies establish that the dosing interval significantly impacts the immunogenicity and protective efficacy of plasmid DNA vaccines.     

Nasal Immunization of Mice with Human Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing Antibodies in Mucosal Secretions

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 μg of VLP given at weekly intervals to anesthetized mice induced high (>10⁴) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-μg VLP systemic priming followed by two 5-μg VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.     

Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla

Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids--inactivated extracellular products--from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide [LPS]) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion). To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activity in mucus and serum. To evaluate protection, we calculated the relative percent survival (RPS) after intraperitoneal (i.p.) injection and bath challenge of the pathogen. The overall results indicate that: (1) capsular antigens seem to be essential for protective immunization; (2) vaccines confer the highest protection when administered by i.p. injection; (3) booster is needed to achieve good protection by immersion; (4) enriching the vaccine with toxoids enhances protection to optimal levels (RPS values around 70 to 100%, depending on the delivery route); and (5) the protective effect in serum and mucus depends on the route of administration and seems to be related to the production of specific antibodies.     

An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.