Intranasal immunization with Chlamydia trachomatis, serovar E, protects from a subsequent vaginal challenge with the homologous serovar.

To vaccinate against a vaginal challenge with Chlamydia trachomatis, C3H/HeJ (H-2k) mice were immunized intranasally (i.n.) or intraperitoneally (i.p.) with 1 x 10(6) inclusion forming units (IFU) of C. trachomatis, serovar E and i.n. with 1 x 10(6) UV inactivated IFU of serovar E. Animals inoculated i.n. with mock infected HeLa 229 cells were used as controls. Upon a vaginal challenge with 5 x 10(3) IFU of serovar E, mice immunized i.n. with viable serovar E exhibited significant protection as judged by the number of mice infected compared to controls (p < 0.05). In contrast, mice immunized i.n. with serovar E that had been UV-inactivated, were not protected from a subsequent vaginal challenge with serovar E. Mice immunized i.p. with serovar E showed attenuation of the infection by 4 weeks after challenge compared to control mice as to the number of animals with positive vaginal cultures (p < 0.05). Of the immune parameters examined, the best correlation with protection was seen with Chlamydia specific IgG and IgA vaginal titers and lymphoproliferative responses to serovar E. In summary, mucosal immunization with viable serovar E partially protected mice against a subsequent vaginal challenge, thereby showing that it is possible to elicit a protective response to a human strain of C. trachomatis at a distant mucosal site in this animal model.     

Mucosal immunization with recombinant MOMP genetically linked with modified cholera toxin confers protection against Chlamydia trachomatis infection

Chlamydia trachomatis is a major human health pathogen due to its role in sexually transmitted diseases. Thus, there is a need to develop an effective vaccine at the mucosal surface against this pathogen. In an effort to develop a mucosal vaccine, a modified cholera toxin gene was genetically linked to the C. trachomatis MoPn NiggII MOMP gene to generate a recombinant protein with the mucosal adjuvant properties of the cholera toxin and immunological antigenicity of the chlamydial protein. The recombinant fusion protein (rMOMP) was expressed in E. coli, purified and analyzed by SDS-PAGE, immunoblot, and GM1-ELISA, and subsequently used to immunize BALB/c mice via intranasal (i.n.) and intravaginal (vag.) routes. The rMOMP protein administered via the i.n. route induced a higher concentration of anti-MOMP specific antibodies in both serum and vaginal washes as compared to mice immunized with Chlamydia or PBS. Antibody isotype analysis revealed that i.n. administration of rMOMP to mice induced higher concentrations of serum and vaginal wash IgA, IgG1, IgG2a, and IgG2b antibodies. Vaginal washes from all immunized mice following a chlamydial challenge infection were analyzed by indirect immunoflourescence to study the level of protection provided by various immunogens. Maximum protection against C. trachomatis as assessed by reduction in C. trachomatis inclusion forming units (IFU) was provided by i.n. immunization of mice with rMOMP. This is a first report using genetic fusion of cholera toxin and MOMP genes and provides a novel approach for the design and development of a mucosal vaccine against Chlamydia.     

Experimental Chlamydia pneumoniae infection in NIH/S mice: effect of reinoculation with chlamydial or cell preparation on culture,PCR and histological findings of lung tissue

The cellular components present in chlamydial preparations may contribute to the course of the experimental infection. NIH/S mice were inoculated and reinoculated intranasally with Chlamydia pneumoniae or a cellular preparation. The mock inoculation induced only mild histological changes in the lungs, which possibly induced partial protection against subsequent C. pneumoniae infection and, when given as reinoculation, possibly reactivated the culture-negative infection as culture-positive. In addition, serum antibodies against mouse heat shock protein 60 (Hsp60) were found in a few mice. In conclusion, the main immunopathogenic factors in a C. pneumoniae mouse model are chlamydial components. However, a cellular preparation may participate in an inflammatory reaction. Autoimmunity against Hsp60 may also play a role in the pathogenesis of C. pneumoniae infection.     

Serological markers of Chlamydia pneumoniae,cytomegalovirus and Helicobacter pylori infection in diabetic and non-diabetic patients with unstable angina pectoris

The possible role of inflammation in coronary artery disease (CAD) is being recognised, while markers of inflammation (e.g., CRP) and infection with Chlamydia pneumoniae (C. pneumoniae), cytomegalovirus (CMV) and Helicobacter pylori (H. pylori) have been proposed as risk factors for CAD. However, these associations require further evaluation. It is a known fact that diabetic patients suffer from impaired immune response to some pathogens and a high incidence of atherosclerosis. In this case-control study we investigated serological markers of infection with C. pneumoniae, CMV, and H. pylori in a group of 140 patients with unstable angina pectoris (UA), 52 of them having type 2 diabetes mellitus, and in a matched control group. Anamnestic (IgG) and acute infection (IgA) antibodies against the above agents were tested using ELISA or indirect immunofluorescence tests. In patients with UA we found a significantly higher seroprevalence and titres of IgG antibodies against C. pneumoniae (p = 0.04) and increased titres of IgG antibodies against CMV (p = 0.007). No differences were found in IgA antibody response to these pathogens. Antibody response to H. pylori was similar in both groups tested. In diabetic patients with UA, the frequency of group-common IgG antibodies against C. pneumoniae was higher than in the non-diabetic UA patients. The other serological markers studied were comparable in the patients with or without diabetes mellitus. Our findings confirmed association of C. pneumoniae and CMV with cardiovascular heart disease. Moreover, diabetes mellitus may predispose the patients to C. pneumoniae infection. However, serological markers observed do not indicate that destabilisation of angina pectoris is associated with acute C. pneumoniae or CMV infection. No relationship was found between UA and H. pylori infection.     

肺炎衣原体感染对巨噬细胞内ox-LDL源性脂质蓄积的影响

目的 研究肺炎衣原体(Chlamydia pneumoniae,Cpn)感染对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)处理RAW264.7后细胞内脂质蓄积的影响.方法 Cpn在Hep-2细胞内增殖,用RAW264.7进行实验.Giemsa染色检测RAW264.7内Cpn感染情况,油红O染色检测细胞内脂质摄取情况,RT-PCR检测ABCA1mRNA表达情况.结果 油红O染色见RAW264.7内脂质摄取增加,RT-PCR检测ABCA1mRNA的表达增加.结论 Cpn感染促使RAW264.7内ox-LDL源性脂质蓄积增加,在此过程中ABCA1mRNA的表达增加,为进一步阐明肺炎衣原体感染和动脉粥样硬化关系提供了实验依据.     

Effect of intragastric and intraperitoneal immunisation with attenuated and wild-type LACK-expressing Listeria monocytogenes on control of murine Leishmania major infection

Stable chromosomal constructs of attenuated DeltaactA and wild-type Listeria monocytogenes expressing the Leishmania major protein LACK were tested as live vaccine vectors in the Th2-orientated chronic L. major murine infection model. These vectors, either by intraperitoneal (i.p.) or intragastric (i.g.) route, were able to induce a strong CD4 Th1 immune response that was correlated with slower parasite growth in the infected footpad. Significant protection against L. major infection was observed in BALB/c mice, ranging from delay in the lesion onset to full protection in 80% of the challenged animals, depending on the size of the parasite inoculum challenge. The i.g. route gave clinically higher protection level than the i.p. route. Both bacterial vectors were as efficient, suggesting that the extent of in vivo bacterial dissemination and multiplication did not seem to be a key parameter for induction of an efficient protective immune response.     

A study of human respiratory tract chlamydial infections in Cambridgeshire 1986-88

Human respiratory tract chlamydial infections have been studied in Cambridgeshire for many years, but until recently we have been unable to distinguish between infection with Chlamydia psittaci or Chlamydia pneumoniae (TWAR). In this study, we have employed the micro-immunofluorescence (micro-IF) test for this purpose and to look for the relative incidence of C. psittaci and C. pneumoniae infections in Cambridgeshire. Among 50 patients with community-acquired respiratory tract symptoms whose serum samples had Chlamydia complement fixation test titres greater than or equal to 64, 25 had evidence of recent C. psittaci or C. pneumoniae infection. Nineteen (76%) of the 25 patients had evidence of recent C. psittaci infection and of these 16 (84%) had recently had contact with birds. Six patients (24%) had evidence of recent C. pneumoniae infection, and of these, only two (33%) had recently had contact with birds. While C. psittaci was grown from several of the birds associated with human C. psittaci infection, it was not cultured from any of the birds in contact with the two human C. pneumoniae cases.     

DNA immunization followed by a viral vector booster in a Chlamydia pneumoniae mouse model

Vaccination against Chlamydia pneumoniae would be a beneficial strategy for either preventing or controlling infection by this human respiratory pathogen that also causes persistent infections. In the present study, we used recombinant Semliki Forest virus (rSFV) particles for delivering C. pneumoniae antigens major outer membrane protein (MOMP) or outer membrane protein 2 (Omp2) to the mice or applied the prime-boost technique, where mice were first primed with naked DNA and then boosted with the viral vector coding for the same proteins. Partial protection suggested by the reduced number of cultivable bacteria from the lungs of the challenged mice was seen in mice immunized by either method with MOMP expressing constructs. A significant protection was also achieved after DNA/rSFV immunization with Omp2. DNA priming followed by rSFV boosting induced a more prominent IFN-gamma production after challenge at the site of the infection in pulmonary and mediastinal cells.     

灰仓鼠作为多房棘球蚴感染实验动物模型的研究

目的 :研究多房棘球蚴 (Em)感染对灰仓鼠的影响 ,进一步确定灰仓鼠作为Em感染实验动物模型的价值。方法 :对灰仓鼠感染Em后包囊的生长、感染鼠的繁殖及后代再感染进行观察研究。结果 :该鼠感染后 5及 7个月包囊重量从 6.60g增加至 14 .46g ,差异具有极显著性。感染后 4个半月以内 ,灰仓鼠可以继续正常繁殖而不影响包囊生长。繁殖的后代对Em再感染无遗传免疫性。结论 :灰仓鼠对Em感染敏感性高 ,囊泡发育佳、生长速度快 ,加之其体型小、适应性强、耐受性好、易饲养、易繁殖等优点 ,是Em感染理想的实验动物模型。     

肾综合征出血热病毒呼吸道感染免疫抑制小鼠的研究

采用肾综合征出血热（ＨＦＲＳ）病毒经呼吸道和腹腔注射感染环磷酰按免疫抑制的成龄ＢＡＬＢ／ｃ小鼠．实验发现ＨＦＲＳ病毒通过两种感染途径均可引起免疫抑制动物发病和死亡，病毒气溶胶通过呼吸道感染引起动物死亡率为４３％，腹腔注射感染引起的死亡率为６１％。该病毒通过呼吸道可以迅速扩散至全身，从肺洗液巨噬细胞可以分离得到病毒．该结果表明，呼吸道是ＨＦＲＳ病毒敏感的侵入途径，在体内的扩散可能是肺巨噬细胞游走携带作用的结果；对于免疫功能低下的动物，呼吸道感染可以导致急性致死性感染。     

Chlamydia pneumoniae genome sequence analysis and identification of HLA-A2-restricted CD8+ T cell epitopes recognized by infection-primed T cells

In the present study, we performed in silico analysis of Chlamydia pneumoniae genome sequence to identify human HLA-A2-restricted T cell epitopes. Thirty-one Chlamydia-specific protein antigens were selected and peptides were derived thereof using an HLA-A2 epitope predictive algorithm. Firstly, we tested binding of 55 selected 9mer peptides to HLA-A2 in vitro. Next, infection of HLA-A2 transgenic mice with C. pneumoniae elementary bodies and assessment of effector CD8+ T cells allowed us to identify which of the epitopes binding to HLA-A2 in vitro were recognized by C. pneumoniae infection-primed CD8+ T cells. Finally, we could confirm that CD8+ T cells in association with HLA-A2 recognized the most reactive peptides when the corresponding full-length genes were used to DNA-immunize HLA-A2 transgenic mice. By using this approach, a novel HLA-A2-restricted epitope in the outer membrane protein A (OmpA) of C. pneumoniae was identified, which proved to mediate specific lysis of peptide-loaded target cells.     

DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlamydial infection from the lower to the upper genital tract in C3H/HeN mice

Chlamydia trachomatis pgp3 DNA immunized (no. 300) and non-immunized (no. 300) C3H/HeN mice were infected by vaginal inoculation with infectious C. trachomatis serotype D elementary bodies (EBs) and the spread of infection to the salpinges was assessed by cell culture isolation from tissue homogenates 7, 14, 21, 28, 35 and 42 days post-infection (p.i.). Overall, the pgp3-DNA immunization prevented salpinx infection in 94 (56%) mice, if compared with the 168 positive animals found among the non-immunized animals (P < 0.001). A group of negative control animals (i.e. mice immunized with plasmid DNA containing an irrelevant insert) was not protected, whereas all the mice of a positive immune control group (mice that had resolved a primary genital C. trachomatis infection) were resistant to re-infection. Pgp3 DNA immunization induced both humoral and mucosal anti-pgp3 antibodies.     

非典型病原体引起下呼吸道感染的调查

目的：了解肺炎支原体和肺炎衣原体在下呼吸道感染率。为经验用药提供依据。方法：双盲法从15所医院收集128位临床诊断疑由肺炎支原体或肺炎衣原体，引起的下呼吸道感染患者在不同病程收集血清标本335份，用固相酶联免疫吸附检测肺炎支原体IgG和IgM，肺炎衣原体IgG和IgM。结果：近期感染：单一肺炎支原体感染患者占16．4％，单一肺炎衣原体感染占17．2％，混合感染患者占17．2％，近期总感染率：50．8％；近期＋既往总感染率：80．5％，其中；肺炎支原体感染患者占49．2％，肺炎衣原体感染占52．3％，成人和儿童的近期感染率分别为41．7％和78．1％，结论：肺炎支原体和肺炎衣原体是社区获得下呼吸道感染的主要病原体。     

Intranasal vaccination of mice against infection with Mycobacterium tuberculosis

The intranasal (i.n.) route of immunisation, has recently been of active interest in endeavours to improve the efficacy of vaccination against a number of respiratory infections. Here, we examined the outcome of tuberculous infection in BALB/c mice. I.n. application of the BCG-Pasteur strain was found to be highly protective against challenge infection with the pathogenic H37Rv strain given after a 4-week interval, reflected by the 100-fold reduction of CFUs in both lungs and spleens. Vaccination with the recombinant PstS-1 antigen and cholera toxin significantly protected against the challenge given 10 days later, but only marginally after 12 weeks. Histological examination showed, that i.n. vaccination abrogated the confluent infiltration of lungs with inflammatory cells, which surrounds the granulomas in H37Rv challenged control mice. In conclusion, the strong protection demonstrated by BCG suggests that the i.n. route of vaccine delivery deserves further attention toward improving vaccination against tuberculosis.     

Chlamydia pneumoniae strain TWAR, Mycoplasma pneumoniae, and viral infections in acute respiratory disease in a university student health clinic population

Clinical and serologic data were collected on 667 University of Washington students who presented to the David Hall Student Health Center between 1983 and 1987 with acute respiratory disease. Sera were tested for evidence of acute or past infections with Chlamydia pneumoniae strain TWAR, Chlamydia trachomatis, Mycoplasma pneumoniae, influenza A virus, influenza B virus, adenovirus, and respiratory syncytial virus. Pharyngeal swab specimens were cultured for C. pneumoniae and C. trachomatis, but not for the other agents. Evidence of acute infection with C. pneumoniae was found in 20 patients and evidence of an acute infection with M. pneumoniae in 29 patients. C. pneumoniae was associated with 9% and M. pneumoniae with 11% of 149 pneumonias diagnosed clinically, and with 20% and 22%, respectively, of the 59 pneumonias confirmed on chest radiograph. There was no evidence of seasonality in C. pneumoniae or M. pneumoniae infections. Compared with patients with M. pneumoniae, patients with C. pneumoniae were less likely to have a temperature greater than 37.8 degrees C (10% vs. 34%), but were more likely to present with a sore throat (80% vs. 52%) or hoarseness (30% vs. 3%). The mean number of days from onset of symptoms until enrollment was longer in patients with C. pneumoniae infections than in those with M. pneumoniae (12.8 vs. 7.9 days), or those with a viral infection (12.8 vs. 7.3 days), suggesting a more gradual onset of disease caused by C. pneumoniae.     

Vaccination of newborn mice induces a strong protective immune response against respiratory and genital challenges with Chlamydia trachomatis

Chlamydia trachomatis infections can occur early in life and may result in long-term sequelae. To assess the feasibility of implementing a vaccine in newborns, groups of 2-day-old BALB/c mice were immunized intranasally (i.n.) with 1x10(4) inclusion forming units (IFU) of C. trachomatis mouse pneumonitis (MoPn). As a control, newborn mice were sham-immunized i.n. with minimal essential medium. In the vaccinated animals, strong Chlamydia-specific humoral and cell-mediated immune responses were observed. Six weeks after immunization, mice were challenged with MoPn i.n. or intravaginally (i.vag.). For the i.n. challenge, mice were inoculated with 10(4) or 10(5)IFU of MoPn per mouse, and in the case of the i.vag. challenge, each animal received 10(6)IFU. By day 10 post-infection (p.i.), the vaccinated mice challenged i.n. with 10(4)IFU, had gained an average of 6.7+/-1% of their body weight. In contrast, the sham-immunized mice had lost 14.9+/-1% of their weight (P<0.05). The mean number of IFU/lungs in the vaccinated animals was 800+/-300, while for the sham-immunized mice was 211+/-49x10(6) (P<0.05). Significant differences between the Chlamydia-vaccinated and the sham-immunized mice were also found in the groups challenged with 10(5)IFU. In the mice challenged i.vag., a significant decrease in the number of mice with positive cultures, and the intensity and duration of vaginal shedding was noted in the vaccinated mice compared to the sham-immunized mice (P<0.05). In conclusion, these results indicate that vaccination of neonatal mice can result in a protective response against a subsequent pulmonary or genital challenge with Chlamydia.     

Immunization with the Chlamydia trachomatis major outer membrane protein,using the outer surface protein A of Borrelia burgdorferi as an adjuvant,can induce protection against a chlamydial genital challenge

Two strains of mice C3H/HeN (H-2(k)) and BALB/c (H-2(d)) were immunized with the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) using the Borrelia burgdorferi outer surface protein A (OspA) as an adjuvant. As a control, groups of mice were inoculated with ovalbumin instead of MOMP. Female mice were immunized using three different routes: intramuscular (i.m.) plus subcutaneous (s.c.), intranasal (i.n.) and perivaginal and perisacral (p.vag.+p.sac.). Significant humoral and cell mediated immune responses developed particularly in mice inoculated by the i.m.+s.c. routes as determined by the levels of chlamydial specific antibody in the serum and genital secretions and a T-cell proliferative assay. Following immunization the animals were challenged in the genital tract with C. trachomatis MoPn and the course of the infection followed by vaginal cultures. Significant protection against infection was achieved in the C3H/HeN mice inoculated i.m.+s.c. with MOMP+OspA, as shown by the intensity and duration of vaginal cultures, and by the number of mice with positive cultures. On the other hand in BALB/c mice there was only a decrease in the number of animals with positive vaginal cultures. Six weeks after the challenge the mice were mated and the outcome of the pregnancy evaluated. In both the C3H/HeN and the BALB/c mice immunized i.m.+s.c. with MOMP+OspA there was significant protection against infertility as shown by the number of animals with bilateral fertility and number of embryos per uterine horn. In conclusion, immunization using C. trachomatis MOMP, and B. burgdorferi OspA as an adjuvant, can induce significant protection against a chlamydial genital challenge.     

四川省首起传染性非典型肺炎病例的病原学及血清学研究

目的：对四川省首起传染性非典型肺炎病例病原学及血清学进行研究。方法：病原分离及血清学检测。结果：细胞及鸡胚培养未分离到相应病原；病例甲、病例乙的SARS冠状病毒IgM和IgG抗体阳性，病例丙的肺炎衣原体IgM抗体阳性。结论：两例患者为SARS冠状病毒感染，另一例可能合并肺炎衣原体感染。     

Immunization with the Chlamydia trachomatis major outer membrane protein, using adjuvants developed for human vaccines, can induce partial protection in a mouse model against a genital challenge

To test several vaccines for Chlamydia trachomatis we vaccinated BALB/c and C3H/HeN female mice with a purified preparation of the native mouse pneumonitis (MoPn) major outer membrane protein (MOMP). The MOMP was formulated with anyone of three different adjuvants MF59, LT-K63 or LT-R72. As a negative control the animals were immunized with ovalbumin. Positive controls were inoculated intranasally (i.n.) with 10(4) inclusion-forming units (IFU) of C. trachomatis MoPn. High levels of Chlamydia-specific antibodies were detected in the serum and vaginal washes of the mice immunized with MOMP. Using a lymphoproliferative assay (LPA) a significant response was obtained in splenocytes from most of the groups of mice vaccinated with MOMP. Two weeks after the last immunization the mice were challenged in the left ovarian bursa with 10(5) IFU of C. trachomatis MoPn and vaginal cultures were collected for a period of 6 weeks. Overall, BALB/c and C3H/HeN mice immunized with MOMP showed a decrease in the severity and length of the infection but the difference with the controls was not statistically significant. Following mating the percentage of mice with bilateral fertility was not significantly different between mice vaccinated with MOMP and their respective ovalbumin-immunized controls. However, the C3H/HeN mice immunized with MOMP using MF59 or LTR72 as adjuvants had significantly more embryos per mouse than the control groups. In conclusion, mice immunized with native MOMP and adjuvants developed for human vaccines showed significant Chlamydia-specific immune response and a limited protection against infection and long-term sequelae.     

Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease.

The prevalence of chronic Chlamydia pneumoniae infection was assessed in 54 patients with established chronic obstructive pulmonary disease (COPD), 41 of these with severe COPD (group I), 13 with mild to moderate COPD (group II), and in 23 patients with community-acquired pneumonia (controls, group III). Specific IgG and IgA antibody levels and circulating immune complexes (ICs) were measured in paired sera, and specific secretory IgA (sIgA) levels in sputum specimens. A polymerase chain reaction (PCR) test was used for the detection of C. pneumoniae in sputum. According to our definite diagnosis criterion, 65% of the COPD patients showed evidence of suspected chronic C. pneumoniae infection and the prevalence was still higher (71%) in patients with severe disease. The occurrence of specific markers of infection was invariably highest in patients with severe COPD, next-highest in patients with mild to moderate COPD and lowest in pneumonia patients. The association between COPD and C. pneumoniae infection persisted after controlling for the potential confounding factors.