Experimental Salmonellosis X. Cellular Immunity and Its Antibody in Mouse Mononuclear Phagocytes

A cell-associated antibody was detected in the peritoneal mononuclear phagocytes (referred to as monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis, by use of immune transfer and immune adherence hemagglutination techniques. The cellular antibody inhibited the growth of a virulent strain of S. enteritidis with the aid of complement and lysozyme on nutrient agar plates. This type of bactericidal antibody could not be detected in the monocytes of mice immunized with killed vaccine of S. enteritidis. The antibody extracted from the peritoneal monocytes of mice hyperimmunized with live vaccine was identified as a macroglobulin by ultracentrifugal analysis.     

以小鼠检测鸡霍乱G190E_（40）疫苗的效力试验

为检测鸡霍乱Ｇ１９０Ｅ４０疫苗,以该苗的不同剂量接种小鼠,测出其最小免疫量和安全性。并以同法对鸡进行平行试验。之后分别以Ｃ４８—１强毒的最小致死量攻击。比较此法对两种动物的差异。试验结果表明,用小鼠替代鸡检测鸡霍乱苗,即安全,又可降低检测成本,因此本法具有实际应用价值。     

The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein

The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.     

A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.     

The experimental validation of the advantages of combined emergency (fluoroquinolones) and specific (EV Nalr) prevention of plague versus their sequential use]

Mice immunization with reference vaccine at the early stage of plague infection provided animals survival and prolonged mean survival period up to 2-5 days. Ciprofloxacin, ofloxacin and pefloxacin prevents development of post vaccine immunity at white mice, immunized by reference vaccine strain EV. Nalidixic acid and norfloxacin effect on post vaccine immunity was lower. Use of immunogenic strain EV Nafr (resistant to nalidixic acid and fluoroquinolones) provided antiplague immunity formation at the background of fluoroquinolones prophylaxis. Ciprofloxacin, ofloxacin and pefloxacin used for plague prophylaxis at white mice infected with Yersinia pestis (about 1000 LD50) inhibited postinfective immunity development. Nalidixic acid and norfloxacin didn't demonstrate such effect. Urgent (fluoroquinolones) and specific (EV Nalr) combined prophylaxis was evaluated as more effective for a 5-day period and provided the development of antiplague immunity.     

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice

Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 microgram to 1000 micrograms. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.     

The new vaccine strains (or variants) of Francisella tularensis

Abstract A colony (N83) of the vaccine strain of Francisella tularensis (15/10) and a strain (N268) isolated from a water sample in nature were revealed for susceptibility to cultivation at 42°C. Both strains had low virulence for white mice and were avirulent for guinea pigs but possessed high immunogenicity in these animals. The spontaneous mutant of vaccine strain 15/10 showed resistance to doxicycline and rifmanpicine (15/10 Dox ^r40 Rif ^r40). The obtained mutant had biological characteristics similar to the parent vaccine strain. It provided immunity in experimental animals when vaccination and antimicrobial agents were used in combination.     

Evaluation of the immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins

Immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins were under study. The immunological safety of the vaccine was evaluated by the absence of antibodies to denaturated and native DNA (d-DNA and n-DNA). As shown with the use of the enzyme immunoassay (EIA), the administration of the vaccine did not induce antibody formation to d-DNA and n-DNA during the observation period. The titer of bactericidal antibodies in the immune bacteriolysis assay (IBA) to the vaccine strain B:2b:P1.2 after immunization increased four-fold and greater in 80% of the vaccinated persons. The significant increase of bactericidal antibodies to heterologous strains B:2a:P1.2 and B:15:P1.7 was registered in 20-30% of the vaccinees, respectively. A month after the repeated vaccination an increase in specific IgG antibodies to the complex antigen was found to occur according to EIA results. The use of RIB made it possible to evaluate the preventive activity of group B meningococcal vaccine as a whole and to suppose that the vaccine induced mainly type-specific response.     

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice

Abstract Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 μg to 1000 μg. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.     

Effect of pertussis vaccine on the functional activity of the peritoneal and splenic macrophages in 2 mouse strains genetically differing by the intensity of their immune response

The immunostimulating effect of corpuscular pertussis vaccine on the antigen-presenting and bactericidal functions of peritoneal and splenic macrophages in CBA and C57BL/6 mice, differing in the intensity of immune response to sheep red blood cells and Salmonella typhimurium, has been studied. The study has revealed that the injection of pertussis vaccine alters the functional activity of the cells under study, the effect depending on the immunizing dose, the strain of mice and the time elapsed from the moment of immunization. Pertussis vaccine enhances the low capacity of macrophages for antigen presentation in C57BL/6 mice with low responsiveness and alters the resistance of peritoneal and splenic macrophages to the cytopathic action of salmonellae.     

Comparative evaluation of the immunological activity of new typhoid vaccines with established prophylactic effectiveness (data from a controlled experiment)]

The authors present the results of studying the immunological efficacy of a dry alcoholic typhoid vaccine enriched with S. typhi Vi-antigen in the assessment of this vaccine in controlled epidemiological trial during the immunization of children aged 7--8 years. O- and Vi-antibodies were tested in the reaction of hemagglutination, H-antibodies--in the agglutination reaction with the microbial diagnostic agent, the properties of antibodies--in a test with cystein, and bactericidal properties of the sera--against the virulent S. typhi strain. Examination of 355 coupled sera obtained before and 3 weeks after the immunization demonstrated a high level of Vi-(1:47) and of the O-(1:580) antibodies and high bactericidal properties of the sera in persons vaccinated with the alcoholic vaccine enriched with the S. typhi Vi-antigen. The results obtained and the data on the formation of prolonged immunity following a simgle immunization suggested that a high protective effect was caused by a combined action of the O- and Vi-antigens contained in the vaccine in optimal doses.     

Effect of Mesua ferrea Linn. flower extract on Salmonella

Based on its traditional uses in folk medicine, the whole flower extract of Mesua ferrea Linn. was tested for its in vitro antimicrobial efficacy against five different strains of Salmonella spp. All the strains were found to be highly sensitive to the extract, MIC of the extract against each organism being 50 microg/ml. The extract was tested in vitro for its mode of antibacterial activity against S. Typhimurium NCTC 74 and it was found to be bactericidal in action. In vivo studies of this extract offered significant protection to Swiss albino mice at doses approximately 2 and 4 mg/mouse when challenged with 50 median lethal dose of S. Typhimurium NCTC 74. Further, the extract caused statistically significant reduction in viable count of the strain in liver, spleen and heart blood of challenged mice.     

Formalin-inactivated Venezuelan Equine Encephalomyelitis (Trinidad Strain) Vaccine Produced in Rolling-Bottle Cultures of Chicken Embryo Cells

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus grown in rolling-bottle cultures of chicken embryo cells. Trinidad strain virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin (USP) and antibiotics. Manipulation of multiplicity of inoculum (0.06 to 0.00006) and maintenance medium volume (100 to 300 ml) resulted in high-titered virus yields and only moderate cell destruction when fluids from infected cultures were harvested at 18 to 24 hr. The virus was inactivated at 37 C by 0.05% Formalin within 8 to 10 hr and with 0.1% Formalin within 6 to 8 hr. Single dose, antigen extinction tests in mice performed with 30 small-scale vaccine lots showed excellent potency at either Formalin concentration with inactivation periods ranging from 24 to 96 hr.     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination

Certain pathogens recruit host complement inhibitors such as factor H (fH) to evade the immune system. Microbial complement inhibitor-binding molecules can be promising vaccine targets by eliciting Abs that neutralize this microbial defense mechanism. One such Ag, meningococcal factor H-binding protein (fHbp), was used in clinical trials before the protein was discovered to bind fH. The potential effect of fH binding on vaccine immunogenicity had not been assessed in experimental animals because fHbp binds human fH specifically. In this study, we developed a human fH transgenic mouse model. Transgenic mice immunized with fHbp vaccine had 4- to 8-fold lower serum bactericidal Ab responses than those of control mice whose native fH did not bind the vaccine. In contrast, Ab responses were unimpaired in transgenic mice immunized with a control meningococcal group C polysaccharide-protein conjugate vaccine. In transgenic mice, immunization with an fH nonbinding mutant of fHbp elicited Abs with higher bactericidal activity than that of fHbp vaccination itself. Abs elicited by the mutant fHbp more effectively blocked fH binding to wild-type fHbp than Abs elicited by fHbp that bound fH. Thus, a mutant fHbp vaccine that does not bind fH but that retains immunogenicity is predicted to be superior in humans to an fHbp vaccine that binds human fH. In the case of mutant fHbp vaccination, the resultant Ab responses may be directed more at epitopes in or near the fH binding site, which result in greater complement-mediated serum bactericidal activity; these epitopes may be obscured when human fH is bound to the wild-type fHbp vaccine.     

诃子水提物对泛耐药鲍曼不动杆菌的抑菌作用

摘要：目的 研究诃子水提物对泛耐药鲍曼不动杆菌（PDR-Ab）的抑菌作用。方法 采用二倍稀释法测定诃子水提物对临床分离的泛耐药鲍曼不动杆菌的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。小鼠腹腔注射上述PDR-Ab,建立急性肺炎模型。于造模前4天至造模后2天对小鼠连续灌胃给药,考察小鼠的死亡率、血液白细胞计数及肺组织病理变化。结果 体外实验表明诃子水提物能抑制PDR-Ab在液体培养基中的生长,MIC和MBC分别为0.75 mg/mL和12 mg/mL。体内实验表明,剂量为150 mg/kg诃子水提物灌胃给药能明显降低PDR-Ab感染性急性肺炎小鼠的死亡率,使血液白细胞计数较快恢复,肺组织病理改善明显。结论 诃子水提物对PDR-Ab在体内外均具有明显的抑菌作用,有望用于PDR-Ab感染性疾病的治疗。     

Comparison of meningococcal outer membrane protein vaccines solubilized with detergent or C polysaccharide

Outer membrane proteins (OMPs) were isolated from meningococcal strain H44/76 (B:15:P1.16) by detergent extraction of bacteria. A final product containing class 1 (P1.16), 3(15), 4 OMPs and 5% (w/w) lipooligosaccharide was obtained. Two experimental vaccines were prepared: OMP-detergent and OMP-C polysaccharide. The OMP-detergent vaccine tended to show a better bactericidal: ELISA ratio for the antibodies induced as compared to the OMP-C polysaccharide vaccine. The vaccine induced bactericidal antibodies appeared for the greater part to be directed against the class 1 OMP (P1.16). By comparison of cultures grown in Mueller Hinton Broth with and without 0,25% (w/v) glucose, it was found that monoclonal antibodies against the serotype OMP (class 2 or 3) were not bactericidal against meningococci grown in MHB without glucose. Antibodies against class 1 OMP and lipooligosaccharide were not influenced by this. A new major outer membrane protein (appr. 40kd) is described that may function as a cation-specific porin.     

Effect of a booster dose of serogroup B meningococcal vaccine on antibody response to Neisseria meningitidis in mice vaccinated with different immunization schedules

The generation and maintenance of memory antibody response by different primary immunization schedules with the Cuban-produced outer membrane protein based vaccine was investigated in a murine model. We analyzed the duration of the antibody response (IgG-ELISA and bactericidal titer) and the effect of a booster dose on the antibody response. The IgG avidity index was determined in an attempt to find a marker for memory development. This study also included an analysis of IgG subclasses induced by primary and booster immunization. The specificity of bactericidal antibodies was investigated using local strains of the same serotype/serosubtype (4,7:P1.19,15) as the vaccine strain and mutant strains lacking major outer membrane proteins. A significant recall response was induced by a booster dose given 7 months after a primary series of 2, 3 or 4 doses of vaccine. The primary antibody response showed a positive dose-effect. In contrast, a negative dose-effect was found on the booster bactericidal antibody response. There was a significant increase in IgG1 levels after the fourth and booster doses. Three doses of vaccine were required to induce a significant increase in IgG avidity. Two injections of vaccine induced a significant antibody response to PorA protein, while 4 injections induced a larger range of specificities.     

Early antibody response in mice to either infection or immunization with Salmonella typhimurium

After immunization with either live or heat-killed Salmonella typhimurium, mice responded with an extremely rapid production of bactericidal antibody which was correlated with the appearance of immunity to a heavy challenge dose (100 ld(50)) of the virulent bacteria. Inactivation of sera with mercaptoethanol along with Sephadex fractionation indicated that the observed bactericidal activity was associated with a macroglobulin which was completely mercaptoethanol-sensitive. The unexpected finding, that a heat-killed vaccine gave excellent protection from a challenge dose which killed all unimmunized control mice, seriously challenges the theory attributing immunity against typhoid infection entirely to a cellular host factor produced only in response to a live vaccine.     

Antibody Response and Protection Induced by Immunization with Smooth and Rough Strains in Experimental Salmonellosis

The antibody response of mice to a smooth strain of Salmonella typhimurium was shown previously to be extremely rapid and potent. As measured by the complement-mediated bactericidal reaction, it was also found to be highly specific as well as reproducible. Experiments which studied the effects of antigen type (live or heat-killed), antigen dose, and the route of immunization indicated that the most rapid and highest antibody response was achieved with live, smooth organisms injected by the intraperitoneal route. Living vaccines of rough strains of either S. typhimurium or S. enteritidis induced antibodies directed against the corresponding smooth organisms. The response to the rough strains was apparently due to antibody production rather than to the simple release of preformed natural antibody. The duration of protection conferred by the rough strain vaccines was closely correlated with the endotoxic content of the immunizing strain. Smooth heat-killed vaccines and a rough live vaccine protected against homologous but not heterologous challenge. In contrast, immunization with a smooth live vaccine protected mice against both homologous and heterologous challenge infections. Protection was not due to a local effect in the peritoneal cavity, since mice were also protected against subcutaneous challenge. The secondary antibody response, induced in immunized animals by the virulent challenge infection, was demonstrated to be rapid and potent, and hence a factor to be considered in protection.