Identification of polymorphic outer membrane proteins of Chlamydia psittaci 6BC

The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.     

Identification of Two Novel Genes Encoding 97- to 99-Kilodalton Outer Membrane Proteins of Chlamydia pneumoniae

Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy showed that epitopes of Omp4 were exposed on the surface of C. pneumoniae elementary bodies, reticulate bodies, and outer membrane complex. Proteins encoded by the C. pneumoniae gene family seem to be dominant antigens in experimentally infected mice.     

Chlamydia trachomatis IncA Is Localized to the Inclusion Membrane and Is Recognized by Antisera from Infected Humans and Primates

Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.     

Identification of genus-specific epitopes on the outer membrane complexes of Chlamydia trachomatis and Chlamydia psittaci immunotypes 1 and 2.

Polyclonal and monoclonal antibodies were used to study the immunogenic and antigenic characteristics of chlamydiae. We focused on the most predominant proteins in the outer membrane complex, the major outer membrane protein (MOMP) and the doublet consisting of proteins of 57 and 62 kilodaltons (57-62 kDa doublet). Immunoblot analyses were performed with chlamydial elementary bodies by using (i) immune sera from sheep which had undergone a recent episode of abortion due to the ovine abortion (OA) strain of C. psittaci, (ii) rabbit hyperimmune anti-C. psittaci (OA) and -C. trachomatis sera, and (iii) monoclonal antibodies to the MOMP of C. trachomatis. The typical pattern of response with polyclonal antisera against heterologous elementary bodies was reactivity with the 57-62 kDa doublet and lipopolysaccharide with weak and sometimes no anti-MOMP activity. Three distinct genus-specific anti-C. trachomatis MOMP monoclonal antibodies showed different patterns of reactivity with the MOMPs of the two immunotypes of C. psittaci and C. trachomatis serovars. Our data confirm the predominance of a genus-specific 57-62 kDa doublet response despite the presence of genus-specific epitopes on the MOMP.     

High-Level Expression of Chlamydia psittaci Major Outer Membrane Protein in COS Cells and in Skeletal Muscles of Turkeys

The omp1 genes encoding the major outer membrane proteins (MOMPs) of avian Chlamydia psittaci serovar A and D strains were cloned and sequenced. The nucleotide sequences of the avian C. psittaci serovar A and D MOMP genes were found to be 98.9 and 87.8% identical, respectively, to that of the avian C. psittaci serovar A strain 6BC, 84.6 and 99.8% identical to that of the avian C. psittaci serovar D strain NJ1, 79.1 and 81.1% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, 60.9 and 62.5% identical to that of the Chlamydia trachomatis L2 strain, and 57.5 and 60.4% identical to that of the Chlamydia pneumoniae IOL-207 strain. The serovar A or D MOMPs were cloned in the mammalian expression plasmid pcDNA1. When pcDNA1/MOMP A or pcDNA1/MOMP D was introduced into COS7 cells, a 40-kDa protein that was identical in size, antigenicity, and electrophoretic mobility to native MOMP was produced. Recombinant MOMP (rMOMP) was located in the cytoplasm of transfected COS7 cells as well as in the plasma membrane and was immunoaccessible. Intramuscular administration of pcDNA1/MOMP in specific-pathogen-free turkeys resulted in local expression of rMOMP in its native conformation, after which anti-MOMP antibodies appeared in the serum.     

Antigenic Analysis of the Major Outer Membrane Protein of Chlamydia spp

The major outer membrane proteins (MOMPs) of several Chlamydia trachomatis serotypes (B, D, G, H, and L2) and of the C. psittaci meningopneumonitis strain were purified by preparatory sodium dodecyl sulfate-(SDS)-polyacrylamide gel electrophoresis. The isolated SDS-polypeptide complexes, which varied in their apparent subunit molecular weights, were used as immunogens to raise hyperimmune rabbit antisera. The specificities of these antisera were determined both by rocket immunoelectrophoresis with the soluble SDS-polypeptide complex as antigen and by micro-immunofluorescence with whole organisms. By rocket immunoelectrophoresis, each of the soluble C. trachomatis MOMPs was immunologically related; however, no immunological cross-reactions occurred with the C. psittaci meningopneumonitis polypeptide, indicating that the MOMPs are antigenically distinct among members of these two chlamydial species. The same antisera were highly reactive with intact organisms by micro-immunfluorescence, demonstrating that at least some of the antibodies raised with SDS-polypeptide complexes reacted with native antigenic sites of these surface proteins. By micro-immunofluorescence, anti-MOMP sera remained species specific; but, unlike the results observed by rocket immunoelectrophoresis, distinct differences in the reactivity and specificity of these antisera were observed among C. trachomatis serotypes. C. trachomatis isolates were separated into two distinct serogroups on the basis of their reactivity with anti-MOMP sera. B complex organisms (B, Ba, D, E, F, G, K, L1, L2, and L3) all reacted strongly with anti-MOMP sera of the B, D, G, and L2 serotypes. In contrast, these same antisera were poorly reactive with the C complex serotypes A, C, H, I, and J. Anti-H MOMP serum was the most serospecific, since high-antibody titers were found only against the homologous H serotype organism. These findings indicate that MOMPs of different strains of C. trachomatis are antigenically complex and that antigenic heterogeneity exists among the surface-exposed portions of the protein.     

Antibody Responses to Recombinant Protein Fragments of the Major Outer Membrane Protein and Polymorphic Outer Membrane Protein POMP90 in Chlamydophila abortus-Infected Pregnant Sheep

Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans.     

Attachment of Cell Walls of Chlamydia psittaci to Mouse Fibroblasts (L Cells)

¹⁴C-labeled cell walls of the 6BC strain of Chlamydia psittaci, prepared from intrinsically labeled chlamydial cells by digestion with deoxycholate and trypsin, associated with mouse fibroblasts (L cells) in a manner comparable to that of intact C. psittaci. Almost half of the host cell-associated cell walls were not dissociated by trypsin, suggesting that they had been attached and then ingested. The attachment of cell walls to L cells was inhibited by a number of treatments known to block association of intact C. psittaci with L cells: heating the cell walls for 3 min or reacting them with antiserum against intact C. psittaci, or pretreating the L cells with trypsin or wheat germ agglutinin. Unlike intact cells of C. psittaci, cell walls were not immediately toxic for L cells, and they did not measurably adsorb neutralizing antibody. As revealed by making cell walls from intact C. psittaci labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination, cell walls contained a much smaller number of surface-labeled proteins than did whole chlamydial cells. The most abundant surface-labeled protein was one with an apparent molecular weight of 43,000. In the final step of cell wall preparation, tryptic digestion of deoxycholate-extracted cells, this major surface protein was partially cleaved to a 40,000-dalton product. When the major surface protein (both the 43,000- and 40,000-dalton moieties) was electrophoretically separated from the other cell wall proteins and used to immunize a rabbit, antibodies that neutralized the infectivity of intact C. psittaci were elicited. It was concluded that cell walls retain the ability to associate with L cells in much the same way as do intact cells of C. psittaci, but, despite the simpler structure of cell walls, the element that binds C. psittaci to host cells cannot yet be identified.     

Identification of Immunologically Relevant Proteins of Chlamydophila abortus Using Sera from Experimentally Infected Pregnant Ewes

Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 × 10⁶ inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein.Chlamydophila abortus, an obligate intracellular Gram-negative bacterium, causes enzootic abortion of ewes (EAE), an important disease of sheep (4-6, 28, 36). In the United Kingdom, C. abortus was responsible for 37% of ovine abortions diagnosed between 2000 and 2006 (28). In Scotland, C. abortus was determined as the etiological agent for 32% of submitted cases in which a diagnosis was reached in 2009 (36). In Ireland, in 2008, 10.3% of submitted samples from ovine abortions were positive for C. abortus (6). However, reported figures are likely to be an underestimate, as the optimal sample for diagnosis of EAE is fresh ovine placenta, which is often omitted when samples are submitted for diagnosis.Chlamydophila abortus has a biphasic development cycle. Infectious extracellular elementary bodies (EB) are metabolically inactive and resistant to adverse environmental conditions. EB attach to host cells, invade, and develop into reticulate bodies (RB), which are metabolically active and multiply by binary fission within a specialized intracellular compartment known as an inclusion. Over a period of 48 to 72 h, inclusions grow in size, and RB mature into EB. The expanding inclusion subsequently ruptures the host cell membrane, thus releasing infectious EB to infect other host cells (47).The complete genome sequence of C. abortus S26/3 has been reported (43). Two major antigens have been studied extensively to date: the major outer membrane protein (MOMP) of approximately 39 kDa (12, 14, 53) and the polymorphic outer membrane proteins (POMP), which are expressed with apparent molecular masses of 85, 90, and 105 kDa (12, 19-21, 40, 46, 47, 49). Other antigenic proteins identified using serum from an infected mouse include the small and large cysteine-rich proteins of 12 and 60 kDa, respectively (49), and a 27-kDa antigen that comigrates with a Chlamydia trachomatis macrophage infectivity potentiator (MIP)-like protein (23). The first C. abortus inclusion membrane protein, Inc766, was recently described (48).This study used experimental infection of pregnant ewes with C. abortus to emulate naturally occurring disease processes. Purified EB were separated by one-dimensional (1-D) and 2-D gel electrophoresis and screened for reactivity with ovine sera by immunoblotting to identify antigens expressed during enzootic abortion of ewes.     

MOMP and MIP DNA-loaded bacterial ghosts reduce the severity of lung lesions in mice after Chlamydia psittaci respiratory tract infection

Chlamydia psittaciis a well known zoonotic pathogen that can lead to severe respiratory disease in poultry, pet birds and humans. Development of an effective and safe vaccine would be the most effective way to control C. psittaci infection. In this study, we used bacterial ghosts (BGs) as a delivery vehicle to evaluate the protective effects of major outer membrane protein (MOMP) and macrophage infectivity potentiator (MIP) DNA vaccines in mice. We found that MOMP/MIP DNA-loaded BGs elicited a better immune response than a naked DNA vaccine, giving increased IgG titers, lymphocyte proliferation responses and higher levels of IFN-γ. After challenge infection, MOMP/MIP DNA-loaded BGs-immunized mice showed lower chlamydial load and inflammation pathology in lung tissues. In addition, we found that MOMP and MIP co-immunization or a heterologous prime-boost strategy could induce stronger immune responses and better protective efficacy against C. psittaci infection. Together, the above results suggest that BGs can act as an effective delivery vehicle for C. psittaci DNA vaccines, and co-immunization or heterologous prime-boost strategy can enhance protective efficacy against infection, thereby providing an alternative strategy for the design of vaccines against C. psittaci.     

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Genotyping of Chlamydophila psittaci by Real-Time PCR and High-Resolution Melt Analysis

Human infection with Chlamydophila (Chlamydia) psittaci can lead to psittacosis, a disease that occasionally results in severe pneumonia and other medical complications. C. psittaci is currently grouped into seven avian genotypes: A through F and E/B. Serological testing, outer membrane protein A (ompA) gene sequencing, and restriction fragment length polymorphism analysis are currently used for distinguishing these genotypes. Although accurate, these methods are time-consuming and require multiple confirmatory tests. By targeting the ompA gene, a real-time PCR assay has been developed to rapidly detect and genotype C. psittaci by light-upon-extension chemistry and high-resolution melt analysis. Using this assay, we screened 169 animal specimens; 98 were positive for C. psittaci (71.4% genotype A, 3.1% genotype B, 4.1% genotype E, and 21.4% unable to be typed). This test may provide insight into the distribution of each genotype among specific hosts and provide epidemiological and epizootiological data in human and mammalian/avian cases. This diagnostic assay may also have veterinary applications during chlamydial outbreaks, particularly with respect to identifying the sources and tracking the movements of a particular genotype when multiple animal facilities are affected.     

Genetic Diversity and Antimicrobial Susceptibility of Campylobacter jejuni Isolates Associated with Sheep Abortion in the United States and Great Britain

Campylobacter infection is a leading cause of ovine abortion worldwide. Historically, genetically diverse Campylobacter fetus and Campylobacter jejuni strains have been implicated in such infections, but since 2003 a highly pathogenic, tetracycline-resistant C. jejuni clone (named SA) has become the predominant cause of sheep abortions in the United States. Whether clone SA was present in earlier U.S. abortion isolates (before 2000) and is associated with sheep abortions outside the United States are unknown. Here, we analyzed 54 C. jejuni isolates collected from U.S. sheep abortions at different time periods and compared them with 42 C. jejuni isolates associated with sheep abortion during 2002 to 2008 in Great Britain, using multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and array-based comparative genomic hybridization (CGH). Although clone SA (ST-8) was present in the early U.S. isolates, it was not as tetracycline resistant (19% versus 100%) or predominant (66% versus 91%) as it was in the late U.S isolates. In contrast, C. jejuni isolates from Great Britain were genetically diverse, comprising 19 STs and lacking ST-8. PFGE and CGH analyses of representative strains further confirmed the population structure of the abortion isolates. Notably, the Great Britain isolates were essentially susceptible to most tested antibiotics, including tetracycline, while the late U.S. isolates were universally resistant to this antibiotic, which could be explained by the common use of tetracyclines for control of sheep abortions in the United States but not in Great Britain. These results suggest that the dominance of clone SA in sheep abortions is unique to the United States, and the use of tetracyclines may have facilitated selection of this highly pathogenic clone.     

Human Antibody Response to Individual Outer Membrane Proteins of Haemophilus influenzae Type b

To evaluate the potential of outer membrane proteins of Haemophilus influenzae as a vaccine, sera from 11 healthy persons and from 23 patients convalescing from disease caused by Haemophilus influenzae type b were assayed for antibodies to individual outer membrane proteins of a single type b isolate, strain Eag, by a gel radioimmunoassay. All 23 patients, ranging in age from 2 months to 62 years, with 17 patients being 24 months or less, had antibodies to some of these proteins in their sera (range, antibodies to 4 to 17 proteins per patient). Although the intensity and spectrum of the response varied, all patients had antibodies to one particular outer membrane protein and 19 patients had antibodies to another, with those patients 5 years and older having antibodies to more proteins than did infants (24 months). In the two cases examined, convalescent sera had greater amounts and broader spectra of antibodies than did acute sera. In addition, 10 of 11 healthy subjects not known to have had systemic H. influenzae disease also had antibodies to individual outer membrane proteins, with older children having greater amounts than did their younger siblings and with children showing a different spectrum of response than that for adults. Thus, antibodies to outer membrane proteins are commonly found in humans. Also, these results and those demonstrating that hyperimmune rabbit antisera to strain Eag reacted with each of five type b substrains possessing some different outer membrane proteins indicate considerable cross-reactivity among these proteins. These results encourage continued consideration of outer membrane proteins in a vaccine.     

Emergence of a tetracycline-resistant Campylobacter jejuni clone associated with outbreaks of ovine abortion in the United States

Campylobacter infection is one of the major causes of ovine abortions worldwide. Historically, Campylobacter fetus subsp. fetus was the major cause of Campylobacter-associated abortion in sheep; however, Campylobacter jejuni is increasingly associated with sheep abortions. We examined the species distribution, genotypes, and antimicrobial susceptibilities of abortion-associated Campylobacter isolates obtained from multiple lambing seasons on different farms in Iowa, Idaho, South Dakota, and California. We found that C. jejuni has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in the United States. Most strikingly, the vast majority (66 of 71) of the C. jejuni isolates associated with sheep abortion belong to a single genetic clone, as determined by pulsed-field gel electrophoresis, multilocus sequence typing, and cmp gene (encoding the major outer membrane protein) sequence typing. The in vitro antimicrobial susceptibilities of these isolates to the antibiotics that are routinely used in food animal production were determined using the agar dilution test. All of the 74 isolates were susceptible to tilmicosin, florfenicol, tulathromycin, and enrofloxacin, and 97% were sensitive to tylosin. However, all were resistant to tetracyclines, the only antibiotics currently approved in the United States for the treatment of Campylobacter abortion in sheep. This finding suggests that feeding tetracycline for the prevention of Campylobacter abortions is ineffective and that other antibiotics should be used for the treatment of sheep abortions in the United States. Together, these results indicate that a single tetracycline-resistant C. jejuni clone has emerged as the major cause of Campylobacter-associated sheep abortion in the United States.     

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.     

Immunochemical Properties of the Major Outer Membrane Protein of Vibrio cholerae

Antisera to the major outer membrane protein of Vibrio cholerae (molecular weight, 48,000) raised in rabbits (i) agglutinated several strains of V. cholerae and (ii) immunoprecipitated outer membrane proteins prepared from both the biotypes and serotypes of V. cholerae. Antibodies of all isotypes to the major outer membrane protein were detected in immune human sera by enzyme-linked immunosorbent assay. These results suggest that the major outer membrane protein was the common outer membrane antigen of V. cholerae which was immunogenic in humans.     

Immunization with <Emphasis Type="Italic">Chlamydia psittaci</Emphasis> plasmid-encoded protein CPSIT_p7 induces partial protective immunity against chlamydia lung infection in mice

The present study evaluated the immune-protective efficacy of the Chlamydia psittaci (C. psittaci) plasmid protein CPSIT_p7 and analyzed the potential mechanisms of this protection. The current study used recombinant CPSIT_p7 protein with Freund’s complete adjuvant and Freund’s incomplete adjuvant to vaccinate BALB/c mice. Adjuvants alone or PBS formulated with the same adjuvants was used as negative controls. Mice were intranasally challenged with 10⁵ inclusion-forming units (IFU) of C. psittaci. We found that CPSIT_p7 vaccination significantly decreased the mouse lung chlamydial load, interferon-γ (IFN-γ) level, and pathological injury. This protection correlated well with specific humoral and cellular immune responses against C. psittaci. In vitro or in vivo neutralization of C. psittaci with sera harvested from immunized mice did not reduce the number of recoverable C. psittaci in the infected lungs, but CD4⁺ spleen cells collected from CPSIT_p7-immunized mice significantly decreased the chlamydial load via adoptive transfer to native mice. These results reveal that the protection conferred by CPSIT_p7 is dependent on CD4⁺ T cells.     

Isolation and Characterization of the Outer Membrane of Borrelia hermsii

The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable β-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [³H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.     

Identification and evaluation of a combination of chlamydial antigens to support the diagnosis of severe and invasive Chlamydia trachomatis infections

Chlamydia trachomatis is the most common sexually transmitted organism In Industrialized countries. Nucleic acid amplification testing, using non-invasively collected specimens, is considered to be the method of choice for diagnosis of chlamydial infections of the urethra and the lower genital tract. Serological testing has the potential to circumvent the problem of specimen sampling in invasive C. trachomatis infections of the upper genital tract. However, only a few defined chlamydial antigens have been used in a standardized diagnostic assay format. In this study, we used serological two-dimensional proteomic analysis to broaden the spectrum of diagnostically relevant C. trachomatis proteins. The genes encoding an assortment of already known chlamydial antigens, as well as immunogenic proteins that have not been described before, were cloned, and the recombinant proteins were purified in order to compare their diagnostic usefulness in parallel with a newly developed line immunoassay. With 189 sera collected from patients with and without C. trachomatis infection, recombinant major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), outer membrane protein 2 (OMP2), translocated actin-recruiting protein, and polymorphic membrane protein D (PmpD) showed the highest level of diagnostic sensitivity and specificity. In patients suffering from ascending and invasive C. trachomatis infections, such as pelvic inflammatory disease and lymphogranuloma venereum, the sensitivity reached with these proteins ranged between 71% (PmpD) and 94% (OMP2), and the specificity ranged between 82% (PmpD) and 100% (MOMP and OMP2). Recombinant thio-specific antioxidant peroxidase, ribosomal protein S1 (RpsA) and hypothetical protein 17 showed lower sensitivity but comparably high specificity, ranging from 94% to 100%. The novel line immunoassay based on defined recombinant antigens has promise for improved serodiagnosis in severe and invasive C. trachomatis infections.