Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis.

Two approaches for presentation of a part of the rickettsial outer membrane protein A (OmpA) of Rickettsia rickettsii, namely (1) recombinant Mycobacterium vaccae (rMV) or (2) recombinant DNA vaccine, stimulated protective immunity against a lethal challenge with the closely related bacterium, R. conorii, in mice. After primary immunization with rMV and booster immunization with homologous recombinant protein, 67 and 55% of mice were protected against challenge in two experiments. DNA vaccination with booster recombinant protein immunization protected six out of eight animals from a lethal challenge. Production of IFN-gamma by antigen-exposed T-lymphocytes of DNA vaccine recipients indicated that cellular immunity had been stimulated.

Identification of protective components of two major outer membrane proteins of spotted fever group Rickettsiae.

Fragments representing the genes of the two major outer membrane proteins of spotted fever group rickettsiae (rOmpA and rOmpB) were tested as DNA vaccines. Immunizations with each of three fragments (rompA4999-6710, rompB1550-2738, and rompB2459-4123) conferred a degree of protection on vaccinated mice against virulent rickettsial challenge. Protection was achieved when DNA immunizations were followed by booster immunizations with the homologous recombinant protein. Proliferation and gamma-interferon secretion were detected after in vitro stimulation of lymphocytes from immunized animals with whole Rickettsia conorii antigen. The data validate particular segments of rOmpA and rOmpB as potent immunogens and hence as sources of immunostimulatory elements with specificity for T lymphocytes, which are the key effectors of protective immunity against rickettsial infections.

Comparison of Ehrlichia chaffeensis recombinant proteins for serologic diagnosis of human monocytotropic ehrlichiosis.

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensis proteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.

Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells.

In a murine model of rickettsial disease in which, as in human rickettsioses, endothelial cells are the major target of infection, depletion of IFN-gamma or TNF-alpha converts a sublethal infection into a uniformly fatal disease with overwhelming rickettsial growth and decreased nitric oxide (NO) synthesis. The kinetics of NO production and rickettsial survival and growth were examined on Days 1, 2, and 3 after inoculation of endothelial cells with Rickettsia conorii under four different experimental conditions: (a) no cytokine treatment, (b) treatment with IFN-gamma and TNF-alpha, (c) treatment with cytokines and NG monomethyl-L-arginine, a competitive inhibitor of NO synthesis, and (d) treatment with sodium nitroprusside, a source of NO. Endothelial cells were examined for the presence of inducible nitric oxide synthase mRNA by specific reverse transcriptase-PCR after stimulation with IFN-gamma and TNF-alpha. Cytokine-stimulated and unstimulated rickettsiae-infected endothelial cells were examined by electron microscopy to observe the cellular and rickettsial events. Transformed and diploid mouse endothelial cells stimulated by the combination of recombinant murine IFN-gamma and TNF-alpha killed intracellular Rickettsia conorii by a mechanism that required the synthesis of NO. The antirickettsial effect and NO synthesis were inhibited by treatment of endothelial cells with NG monomethyl-L-arginine. Addition of nitroprusside, which released NO, also exerted a strong antirickettsial effect in the absence of IFN-gamma and TNF-alpha. Endothelial inducible nitric oxide synthase mRNA was detected 4 hours after cytokine stimulation, increased substantially at 8 hours, and decreased to low levels by 72 hours. Ultrastructural evaluation revealed that endothelial cells effected rickettsial killing in association with autophagy. Double membranes of endothelial cell granular endoplasmic reticulum surrounded rickettsiae, which were also observed being destroyed within phagolysosomes. This study demonstrated for the first time that endothelial cells are capable of killing rickettsiae. When stimulated by the combination of IFN-gamma and TNF-alpha, mouse endothelial cells kill Rickettsia conorii by an NO-dependent mechanism. Within the endothelium, NO exerts a rickettsicidal effect.

Sequence analysis of the 190-kDa antigen-encoding gene of Rickettsia conorii (Malish 7 strain).

The complete nucleotide (nt) sequence of the gene (rompA) encoding the 190-kDa immunodominant surface antigen (rOmpA) of Rickettsia conorii (Malish 7 strain) was determined. Sequence analysis revealed an ORF of 6063 nt encoding a deduced protein of 203,247 Da. Ten consecutive highly conserved repeat units, located at the 5' end of rompA, spanned 2.2 kb. Two types of repeats could be identified: type I of 225 bp, and type II of 216 bp. The order and number of repeats differed from those reported for R. conorii (Kenya tick typhus strain), R. akari (Kaplan strain) and R. rickettsii (R strain). Alignment of the R. conorii (Malish 7 strain) rompA gene with its R. rickettsii homolog revealed 95% nt sequence similarity. The conservation of rompA across several pathogenic spotted fever group (SFG) rickettsial species suggests that it may be a potential candidate for use as a subunit vaccine for SFG rickettsial diseases.