Chemokine Receptor 7 Is Essential for Coxiella burnetii Whole-Cell Vaccine-Induced Cellular Immunity but Dispensable for Vaccine-Mediated Protective Immunity.

BACKGROUND: Protective immunity against Coxiella burnetii infection is conferred by vaccination with virulent (PI-WCV), but not avirulent (PII-WCV) whole-cell inactivated bacterium. The only well-characterized antigenic difference between virulent and avirulent C. burnetii is they have smooth and rough lipopolysaccharide (LPS), respectively. METHODS: Mice were vaccinated with PI-WCV and PII-WCV. Humoral and cellular responses were evaluated using protein chip microarrays and ELISpots, respectively. Dendritic cell (DC) maturation after stimulation with PI-WVC and PII-WVC was evaluated using flow cytometry. Vaccine-challenge studies were performed to validate the importance of the receptor CCR7. RESULTS: Other than specific antibody response to PI-LPS, similar antibody profiles were observed but IgG titers were significantly higher after vaccination with PI-WCV. Furthermore, higher frequency of antigen-specific CD4+ T cells was detected in mice immunized with PI-WCV. PI-WCV-stimulated DCs displayed significantly higher levels of CCR7 and migratory ability to secondary lymphoid organs. Challenge-protection studies in wild-type and CCR7-deficient mice confirmed that CCR7 is critical for PI-WCV-induced cellular immunity. CONCLUSIONS: PI-WVC stimulates protective immunity to C. burnetii in mice through stimulation of migratory behavior in DCs for protective cellular immunity. Additionally, the humoral immune response to LPS is an important component of protective immunity.

Antigenic analysis for vaccines and diagnostics.

Coxiella burnetii infection is frequently unrecognized or misdiagnosed, as symptoms generally mimic an influenza-like illness. However, the disease (Q fever) may result in chronic infection, usually manifesting as potentially fatal endocarditis. The development of a chronic fatigue-like sequela may also occur. Infected ruminants are the major reservoir for infection in humans, primarily through exposure to birth products or aerosols that transmit the bacterium over wide regions. A vaccine against C. burnetii infection has been in use in Australia for abattoir and agricultural workers for many years. The possibility of adverse reactions in those with previous exposure to the agent has prevented its use elsewhere. Subunit vaccines, utilizing chemical extraction of components thought to cause adverse reactions, are in development, but none are yet licensed. Others have sought to combine immunogenic peptides with or without selected lipopolysaccharide components to produce a vaccine without the possibility of adverse reactions. Selected immunogenic proteins have been shown to induce both humoral and cellular immune responses. Although current diagnosis of infection relies on serological testing, the presentation of specific antibody occurs 7-15 days following the onset of symptoms, delaying treatment that may result in prolonged morbidity. PCR detection of DNA to specific C. burnetii antigens in the blood is possible early in infection, but PCR may become negative when PII IgG antibodies appear. PCR is useful for early diagnosis when Q fever is suspected, as in large epidemics, and shortens the delay in the identification of Q fever endocarditis. Others have combined PCR with ELISA or other methods to increase the ability to detect infection at any stage. The search for new diagnostic reagents and vaccines has utilized new methods for discovery of immunoreactive proteins. DNA analysis of the heterogeneity of C. burnetii isolates has led to a greater understanding of the diversity of isolates and a means to determine whether there is a correlation between strain and disease severity. 2-D SDS PAGE of immunogenic proteins reactive with human or animal infection sera and mass spectrometric analysis of specific secreted or outer membrane proteins have identified candidate antigens. Microarrays have allowed the analysis of peptide libraries of open reading frames to evaluate the immunogenicity of complete genomes.

Coxiella burnetii induces apoptosis during early stage infection via a caspase-independent pathway in human monocytic THP-1 cells.

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.

Profiling the humoral immune response of acute and chronic Q fever by protein microarray.

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.

Laboratory maintenance of Coxiella burnetii.

Coxiella burnetii, an obligate intracellular Gram-negative bacterium, is the agent of Q fever, a self-limited flu-like illness that may also present as chronic endocarditis. The ability to persist in the environment at a low infectious dose in aerosols resulted in the classification of C. burnetii as a BSL-3 select agent. Routine propagation of this agent is in embryonated eggs or tissue culture. Purification from host tissues includes multiple differential centrifugations to separate bacteria from host material. Infection of host cells is routinely verified microscopically by using Gimenez stain, fluorescent dyes, or immunofluorescence antibody (IFA) staining. Identification of C. burnetii DNA in host material is measured by PCR. Quantification of purified C. burnetii is accomplished through conversion of optical density to dry weight or, more precisely, by RT-PCR to determine genome equivalents. Serum antibody titer to C. burnetii is determined by microagglutination assay or ELISA.

Mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection in BALB/c mice.

To elucidate the mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection, we compared the protective efficacy and immunogenicity between formalin-inactivated phase I vaccine (PI-V) and phase II vaccine (PII-V) in BALB/c mice. PI-V generated significant protection while PII-V did not confer measurable protection. Analysis of cytokine and subclass Ab responses indicated that both PI-V and PII-V were able to induce a Th1-dominant immune response but did not identify the component of host response that distinguished their ability to induce protective immunity. Interestingly, immunoblot analysis identified a difference between PI-V and PII-V vaccinates in antigenic recognition by specific Ab isotypes. The observation that PI-LPS elicited significant protection but PII-LPS did not confer measurable protection suggests PI-LPS may play a key role in PI-V-induced protection. Adoptive transfer of either immune sera or splenocytes mediated significant protection in naive BALB/c mice, supporting the notion that both humoral and cellular immunity are important for development of protective immunity. However, the evidence that immune sera and B cells were unable to control infection while T cells conferred significant protection in SCID mice supports the hypothesis that T cell-mediated immunity is critical for host defense against C. burnetii infection. This report presents novel evidence to highlight the importance of PI-LPS and Abs in protective immunity and has important implications for the design of new generation vaccines against Q fever.

Identification and characterization of an immunodominant 28-kilodalton Coxiella burnetii outer membrane protein specific to isolates associated with acute disease.

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an approximately 28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a approximately 28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.

Identification and cloning of immunodominant antigens of Coxiella burnetii.

A sublethal-challenge model was established in BALB/c mice by using protection from the development of severe splenomegaly as an indicator of vaccinogenic activity for evaluation of the protective efficacies of vaccine candidates. To determine the immunodominant antigens as defined by reaction to an infection-derived antibody, mouse sera from different stages of experimental infection with various doses of Coxiella burnetii were tested by immunoblotting. Proteins with molecular masses of 14, 16, 21, 28, 32, 45 to 50, 57, and 60 kDa were recognized as immunodominant antigens. Antibody responses in whole-cell antigen (WCA)-vaccinated mice were compared with those in unvaccinated mice by immunoblotting using two-dimensional gel-separated C. burnetii antigens. The results indicated that there were significantly different antibody responses during different stages of vaccination and challenge, suggesting that several specific immunogenic antigens may play critical roles in the protection of mice against challenge. To clone these immunogenic antigens, a genomic DNA library of Nine Mile phase I was screened with convalescent-phase antisera from mice. Eighteen novel immunoreactive proteins with molecular masses ranging from approximately 14 to 67 kDa were cloned and identified. Interestingly, several recombinant proteins reacted with sera from both early-stage infected and WCA-vaccinated prechallenged mice. These results suggest that these proteins may play critical roles in the development of protective immunity and that they are logical candidates for vaccine and serodiagnostic reagents.