Relationship between an 85 kDa protein and the protective effects of Mycoplasma pneumoniae.

In the immunoblot analysis, sera from patients infected with Mycoplasma pneumoniae reacted with the 168 kDa (P1) and the 85 kDa proteins of virulent strain FH-P24 and P24-S1 mutant strain but not with the 85 kDa protein of P24-S11. Sera of hamsters and BALB/c mice, which had been immunized with live vaccines, were tested. In FH-P24 immunized animals, 100% or 80%, and in P24-S1, 40% of hamsters and 60% of BALB/c mice, developed antibodies against the 85 kDa protein, but antibodies were not detected in sera of P24-S11 immunized animals. The correlation between the development of antibodies to 85 kDa protein in the sera of vaccinated animals and the effects of protection by living vaccines were suggested.     

Protective activity of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae

Protective efficacy of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae cultivated on cardiocerebral broth and semisynthetic growth medium respectively was studied in vivo. Fraction with molecular weight 30 - 50 kDa obtained by the method of membrane fractionation had high protective efficacy. Two-dose immunization of mice with this fraction provided 80 - 100% protection from infection by homologous strains of S. pneumoniae and K. pneumoniae. Cross-protective activity of the fraction was revealed when infecting immunized mice by different K-types of K. pneumoniae. Blood sera of mice immunized with 30 - 50 kDa fraction possessed preventive features protecting from infection 90% of animals while 100% of death in the control group. It was determined that protective efficacy of the mentioned fraction was determined by protein-containing antigens because proteolytic disruption of the protein component resulted in loss of protective properties of the preparation.     

Amino acid sequence and antigenicity of the amino-terminus of the 168 kDa adherence protein of Mycoplasma pneumoniae

The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa protein and the involvement of the homologous sequence of the protein in the adherence process. The synthetic peptide when coupled to ovalbumin was immunogenic in rabbits. Antibodies against this peptide epitope could be demonstrated in sera taken during natural M. pneumoniae infection in humans. The structural domain of the 168 kDa protein homologous with the synthetic peptide did not appear to be involved in adherence, as the synthetic peptide or its homologous antibody failed to inhibit adherence of M. pneumoniae.     

Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants

Neonatal mice delivered from mothers preimmunized with heated or formalinized whole cell vaccines of type Ia, Ia/c and III/c group B streptococci were infected with each type of bacteria, and then serum antibodies of mothers and neonates who survived the experiments were measured by enzyme-linked immunosorbent assay. The relationship between the protectivity in neonate mice and the antibody titers to the type specific polysaccharide antigens and the protein c antigen of their sera were examined. In the Ia-immunized group which showed high protection against the type Ia infection, anti-Ia IgG antibody titers were low, and anti-protein c IgG antibody was not detected. Type Ia/c and III/c vaccines were highly effective against both type Ia/c and III/c infection, but less effective in type Ia infection. The protein c antigen was identified in both type strains by the double diffusion assay, and the IgG antibodies to the protein c were significantly high in sera of both maternal mice immunized with types Ia/c or III/c organisms and their newborn infants. High titers of the protein c IgG antibody retained 3 to 4 weeks after the last injection of vaccines which corresponded to the period of pregnancy and lactation. Small amounts of IgM antibody to all antigens were detected only in maternal sera. These results suggest that IgG antibodies to the protein c antigen and to the type-specific polysaccharide antigens are equally important protective factors which are transferable from preimmunized mothers to their newborn infants through placenta and/or lactation.     

Secretion of Cpn0796 from Chlamydia pneumoniae into the host cell cytoplasm by an autotransporter mechanism

By comparison of proteome profiles of purified Chlamydia pneumoniae and whole lysates of C. pneumoniae infected HEp-2 cells, an N-terminal fragment of the previously uncharacterized chlamydial protein Cpn0796 was identified as a secreted protein. A 38 kDa cleavage product of Cpn0796 was present in infected cells, whereas only the 65 kDa full-length Cpn0796 could be detected in purified Chlamydia. Confocal immunofluorescence microscopy demonstrated that Cpn0796 was localized in the Chlamydia membrane in young inclusions. However, at 36 h post infection and later Cpn0796 was detected in the cytoplasm of C. pneumoniae infected HEp-2 and BHK cells. Furthermore, Cpn0796 was detected in the cytoplasm of infected cells in the lungs of C. pneumoniae infected C57Bl mice. When cleavage was inhibited, Cpn0796 was retained in the chlamydiae. We propose that Cpn0796 is an autotransporter the N-terminal of which is translocated to the host cell cytoplasm. This is the first example of secretion of a Chlamydia autotransporter passenger domain into the host cell cytoplasm. Cpn0796 is specific for C. pneumoniae, where five homologous proteins are encoded by clustered genes. None of these five proteins were found to be secreted.     

Parasitological and histopathological effects of immunosuppression in guinea-pigs (Cavia porcellus) experimentally infected with Schistosoma haematobium

The parasitological and histopathological effects of immunosuppression in guinea-pigs (Cavia porcellus) experimentally infected with Schistosoma haematobium were studied. A total of 16 guinea-pigs were divided into four groups (four per group): non-immunosuppressed, non-infected group (NN); immunosuppressed, non-infected group (IN); immunosuppressed, infected group (II); non-immunosuppressed, infected group (NI). The IN and II groups were immunosuppressed with 5?mg/kg prednisolone while the II and NI animals were infected with 200-300 S. haematobium cercariae. Excretion of eggs in urine/faeces, worm burden and histopathology of some vital organs of the guinea-pigs were studied. Eggs of S. haematobium were observed in the urine of the NI and II groups from 9 weeks post-infection and in faeces from 10 and 13 weeks post-infection for the NI and II groups, respectively. However, II animals excreted more viable eggs in urine and faeces than those of the NI group. Worm recovery at 14 weeks post-infection showed that NI and II guinea-pigs had more female worms than male worms and a greater proportion of worm recovery for NI animals was of immature worms. Significant differences (P?0.05). Histological changes, which were notably reactions to adult S. haematobium worms, were observed in the organs of the NI and II groups but these changes were seen more in the organs of the immunosuppressed, infected (II) than in the non-immunosuppressed, infected (NI) guinea-pigs. The results suggest that immunosuppression before infection increased worm survival and had a moderate effect on liver and bladder histology of S. haematobium infected guinea-pigs.     

Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection

Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans. C. pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa. The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C. pneumoniae outer membrane complex. Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response. To address this issue, C57BL/6J mice were infected intranasally with C. pneumoniae and the immune response to primary infection was investigated. We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum. In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay. The responses were shown to be mediated by CD4(+) T cells. To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C. pneumoniae infection.     

The skin-reactive antigens of Mycoplasma pneumoniae.

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the orgnaism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 mug. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform-methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Vaccination of mice with a 30 kDa Schistosoma antigen with and without human adjuvant induces high protection against S. mansoni infection

A 30 kDa antigen was characterized as a hydrophobic polypeptide containing 16 amino acids and evaluated as a potential candidate vaccine against infection by Schistosoma mansoni. CD1 albino mice immunized at 0, 14, and 21 days with 25 or 50 microg of the 30 kDa antigen per mouse with and without alum developed high levels of IgG antibodies (predominantly IgG2a and IgG2b isotypes). When immunized mice were infected with 200 S. mansoni cercariae, the highest protection levels (61% and 65% reduction in worm burden in two separate experiments) were obtained using the 50-microg antigen without alum adjuvant. The granuloma size decreased to 10%, a non-significant level in mice immunized using alum adjuvant. The results demonstrate the ability of the 30 kDa antigen with and without alum adjuvant to protect mice against S. mansoni infection.     

金黄色葡萄球菌LDH多克隆抗体制备及其交叉反应性

目的:在原核载体中表达、纯化金黄色葡萄球菌乳酸脱氢酶,免疫小鼠获得多克隆抗体,使用多克隆抗体分析与其它菌种的交叉反应性。方法:复苏p ET28a-ldh/Bl21重组菌,IPTG诱导重组融合蛋白表达、以抗HIS标签的单克隆抗体进行western-blot鉴定重组蛋白。使用纯化的重组蛋白以及相应的佐剂免疫小鼠,利用ELISA测定血清抗体效价,并使用小鼠抗血清进行免疫印迹法鉴定重组蛋白的反应原性及其与金黄色葡萄球菌、表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌的交叉反应性。结果:SDS-PAGE电泳在39 KDa处可见目的条带,免疫印迹法验证了重组LDH的表达,纯化后获得2.8 mg重组蛋白。纯化蛋白免疫小鼠能诱导产生特异性体液免疫应答,ELISA检测特异性Ig G效价为1:50000,western-blot鉴定显示所制备的多克隆抗血清能分别识别金黄色葡萄球菌重组及天然乳酸脱氢酶,但不识别表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌中天然乳酸脱氢酶。结论:纯化的LDH具有良好的免疫活性,免疫小鼠获得高滴度的多克隆抗体。使用多克隆抗体western-blot显示与其它菌种LDH不存在交叉反应性,为后续使用该重组蛋白进行金黄色葡萄球菌感染的诊断研究奠定基础。     

State of cellular immunity and phagocytes in guinea pigs infected with Mycoplasma pneumoniae

The work presents the results of the study of experimental Mycoplasma pneumoniae infection in guinea-pigs. A considerable increase in the engulfment of mycoplasmas and blood leukocytes was found to occur on days 14-28 after the infection. The correlation between the degree of the engulfment of mycoplasmas by macrophages and the activity of lymphocytes in the reaction of blast-cell transformation with phytohemagglutinin and Mycoplasma antigen was observed. The peculiarities of the course of infection in sensitized animals were revealed. Virulent Mycoplasma pneumoniae strains were found to have a toxic effect on the lymphocytes and macrophages of guinea-pigs. This effect was neutralized by specific antiserum. The importance of these facts for the pathogenesis of Mycoplasma infection is discussed.     

Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome lambda-display library

Streptococcus pneumoniae is a causative agent of otitis media, pneumonia, meningitis and sepsis in humans. For the development of effective vaccines able to prevent pneumococcal infection, characterization of bacterial antigens involved in host immune response is crucial. In order to identify pneumococcal proteins recognized by host antibody response, we created an S. pneumoniae D39 genome library, displayed on lambda bacteriophage. The screening of such a library, with sera either from infected individuals or mice immunized with the S. pneumoniae D39 strain, allowed identification of phage clones carrying S. pneumoniae B-cell epitopes. Epitope-containing fragments within the families of the histidine-triad proteins (PhtE, PhtD), the choline-binding proteins (PspA, CbpD) and zinc metalloproteinase B (ZmpB) were identified. Moreover, library screening also allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the ORF spr0075 in the R6 strain genome sequence. In this work, Spr0075 is first identified as an expressed S. pneumoniae gene product, having an antigenic function during infection.     

Protective immune responses in mice induced by intramuscular and intranasal immunization with a Mycoplasma pneumoniae P1C DNA vaccine

Mycoplasma pneumoniae is an important causative agent of atypical pneumonia. This study was to determine the ability of a DNA expression vector, which encodes the carboxy terminal region of the M. pneumoniae P1 protein (P1C), to induce humoral and cellular immune responses and to protect against M. pneumoniae infection in BALB/c mice. Mice were immunized with pcDNA3.1/P1C by either intramuscular injection (i.m.) or intranasal inoculation (i.n.). Our results showed that p1c DNA immunization generates detectable antibodies specific to M. pneumoniae, and elicits high levels of IgG1, IgG2a, and IgG2b isotypes (P?< 0.01). The levels of IFN-γ and IL-4 in spleen cells of the immunized mice were significantly elevated by immunization via both the i.m. and i.n. methods. Moreover, p1c DNA-immunized mice exhibited detectable protection against M. pneumoniae infection. The lung tissue inflammation was relieved and the histopathologic score (HPS) of pcDNA3.1/P1C-immunized mice was significantly decreased than those in phosphate-buffed saline (PBS) or vaccine-vector-immunized mice (P?< 0.01), whereas there were no significant differences in HPS between i.m. and i.n. vaccination (P?> 0.05). Our results suggest that pcDNA3.1/P1C could be useful for developing a vaccine against M. pneumoniae infection.     

Effect of vaccination against pneumococcal infection and influenza in children with asthma

Assessment of clinical course of asthma and IgG response in children with asthma immunized with pneumococcal polysaccharide vaccine (Pneumo 23) and influenza vaccine (Vaxigrip). 78 children aged 4 - 17 years old were allocated to two groups. Children from the 1st group were immunized against pneumococcal infection and influenza; children from 2nd group were immunized against pneumococcal infection only. Rate of asthma exacerbations in the 1st group of children decreased by 1.7 times compared with the period before vaccination, whereas the same rate in the 2nd group of children decreased by 1.5 times. It was accompanied by the increase of IgG level to antigens of pneumococcalvaccine in blood, which was observed in both groups. Vaccination did not result in increase of IgE levels. Immunization of children with asthma against pneumococcal infection with polysaccharide vaccine or combined immunization against pneumococcal infection and influenza reduced rate of asthma exacerbations and led to formation of immunity to vaccine strains of Streptococcus pneumoniae. Vaccination did not lead to sensitization of children.     

Effect of the Aedes fluviatilis saliva on the development of Plasmodium gallinaceum infection in Gallus (gallus) domesticus

Effect of Aedes fluviatilis saliva on the development of Plasmodium gallinaceum experimental infection in Gallus (gallus) domesticus was studied in distinct aspects. Chickens subcutaneously infected with sporozoites in the presence of the mosquito salivary gland homogenates (SGH) showed higher levels of parasitaemia when compared to those ones that received only the sporozoites. However, the parasitaemia levels were lower among chickens previously immunized by SGH or non-infected mosquito bites compared to the controls, which did not receive saliva. High levels of anti-saliva antibodies were observed in those immunized chickens. Moreover, 53 and 102 kDa saliva proteins were recognized by sera from immunized chickens. After the sporozoite challenge, the chickens also showed significant levels of anti-sporozoite antibodies. However, the ability to generate anti-sporozoites antibodies was not correlated to the saliva immunization. Our results suggest that mosquito saliva components enhance P. gallinaceum parasite development in naive chickens. However, the prior exposure of chickens to salivary components controls the parasitemia levels in infected individuals.     

Three cases of hemolytic Streptococcus infection in guinea-pigs (author's transl)

Three cases of naturally occurring hemolytic streptococcus infection were detected in guinea-pigs which had been delivered from different sources to the Division of Animal Research, Faculty of Medicine, University of Tokyo, Tokyo, during the period from November, 1976, till February, 1977. The clinical manifestations of the infection were differentiated into two types; ie, the acute type with sepsis and pneumonia as main pathologic changes, and the chronic type with abscess formation of submaxillary lymph node. Streptococcus zooepidemicus was isolated from the conjunctiva of about 56% of these guinea-pigs. Healthy guinea-pigs housed with spontaneously infected ones in the same cage suffered from the infection, showing manifestation and pathologic changes similar to the spontaneous cases. Some of them, however, remained apparently healthy for about 2 months harboring the organisms in the conjunctiva or nasal cavity. It is presumed that such carriers become a source of the infection in nature.     

Immune response of chimpanzees infected with human mycoplasmas

Seven chimpanzees were inoculated intra-articularly with one of three Mycoplasma species. Three chimpanzees were inoculated with clinical synovial isolate 1620, and one was inoculated with type strain PG-21 of M. hominis. The fifth animal was inoculated with clinical synovial isolate 2010B, and the sixth was inoculated with the type strain CO of Ureaplasma urealyticum. The seventh animal was inoculated with clinical isolate PI-1428 of M. pneumoniae. The synovial isolates induced intense antibody responses, antibodies recognized more antigens, and the reactive antigens were distinct from the corresponding type strains. Chimpanzees infected with synovial isolate 1620, but not type strain PG-21, recognized antigens of molecular mass 223, 208, 168, 165, 155, 150, 142, 134, 115, 105, 52, 50, 45, 38, and 36 kDa. The chimpanzee infected with synovial isolate 2010B, but not type strain CO, recognized antigens at about 277, 237, 62, 57, and 43 kDa. The major antibody reactive antigens of M. pneumoniae isolate PI-1428 migrated at about 170, 90, 56, 40, 32, and 30 kDa. The reported biologic activities of antigenic proteins derived from these Mycoplasma species are reviewed.     

Transmitted/Founder Simian Immunodeficiency Virus Envelope Sequences in Vesicular Stomatitis and Semliki Forest Virus Vector Immunized Rhesus Macaques

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID₅₀. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.     

Vaccination with DNA vector expressing chlamydial low calcium response protein E (LcrE) against Chlamydophila pneumoniae infection

Chlamydophila pneumoniae is an obligate intracellular human pathogen, which causes acute respiratory tract infections and can also cause chronic infections. C. pneumoniae possess type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and the host cell cytosol. Low calcium response protein E (LcrE) is a part of TTSS. The gene of LcrE in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin, this gene was also cloned into a eukaryotic expression vector (pΔRC). One group of BALB/c mice received an intramuscular pΔRC inoculation then the mice were immunized with purified LcrE protein; the second group of mice was immunized two times with the recombinant plasmid (pΔRCLcrE), and the third group was primed with pΔRCLcrE inoculation then boosted with LcrE protein. LcrE-specific antibody response was induced by DNA immunization with a shift towards Th1 isotype pattern compared to protein-immunization, this shifting pattern was observed in plasmid primed then protein-boosted animals. DNA immunization given as a priming and followed by a protein booster significantly reduced the number of viable bacteria in the lungs after challenge with C. pneumoniae. These results confirm that immunization with pΔRCLcrE can be an effective part of a vaccination schedule against C. pneumoniae.     

Immune responses and protective efficacy induced by 85B antigen and early secreted antigenic target-6 kDa antigen fusion protein secreted by recombinant bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The gamma-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (p<0.05). Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups (p>0.05).