Failure of a heterologous recombinant Sca5/OmpB protein-based vaccine to elicit effective protective immunity against Rickettsia rickettsii infections in C3H/HeN mice

Abstract

Spotted fever group (SFG) rickettsial species are obligate intracellular tick-borne pathogens that are responsible for important human diseases. Previous reports have demonstrated the feasibility of using recombinant surface cell antigen Sca5/OmpB to elicit protective immunity against homologous challenges using murine models of Mediterranean spotted fever and Rocky Mountain spotted fever. In addition, the feasibility of generating cross-protective immunity against related rickettsial species has also been established, but the molecular basis for these phenomena was not explored. Here, we demonstrate that vaccination of C3H/HeN mice with a recombinant OmpB domain derived from Rickettsia conorii induced high titer humoral immune responses that are capable of recognizing the native OmpB protein at the R. rickettsii outer membrane, but this immunization was not sufficient to induce effective protective immunity. In contrast, animals vaccinated with a corresponding OmpB domain derived from R. rickettsii protected animals from fatal outcomes. These results demonstrate that vaccination with nearly identical antigens may not be an effective strategy to induce wide-ranging protective immunity against related SFG Rickettsia species.

Vaccination of animals with nearly identical antigens from closely related rickettsial species may not be an effective strategy to promote wide-ranging protective immunity.

Graphical Abstract Figure.

Vaccination of animals with nearly identical antigens from closely related rickettsial species may not be an effective strategy to promote wide-ranging protective immunity.

INTRODUCTION

The Gram-negative α-proteobacteria of the genus Rickettsia are small (0.3–0.5 × 0.8–1.0 μm), obligate intracellular organisms. Spotted fever group (SFG) rickettsiae including Rickettsia rickettsii (Rocky Mountain spotted fever, RMSF) and R. conorii (Mediterranean spotted fever, MSF) are pathogenic organisms transmitted to mammals through tick salivary contents during the blood meal. MSF, endemic to Southern Europe, North Africa and India, is characterized as a milder rickettsiosis in humans, with 2–3% mortality of reported cases; however, recent accumulating evidence has unveiled that MSF exhibits an expansive geographic distribution, now including central Europe and central and southern Africa, and increased disease severity with mortality rates reported as high as 32% in Portugal in 1997 (de Sousa et al. 2003). RMSF is one of the most severe spotted fever group rickettsioses in the western hemisphere, causing severe morbidity and up to 20% mortality in the absence of appropriate antibiotic therapy (Walker 1989a,b). In the USA, the incidence of RMSF has steadily increased over the last decade with a rate of approximately six cases per million persons in 2010 (http://www.cdc.gov/rmsf/stats/). Whereas the overall case fatality rate in the USA has declined over this period, certain areas such as eastern Arizona have continued to experience approximately 10% fatality rate of individuals with confirmed RMSF diagnosis (http://www.cdc.gov/rmsf/stats/). A re-emergence of Brazilian spotted fever, also caused by R. rickettsii, has also led to a case fatality rate ranging from 30–80% (Toledo et al. 2011; Arguello et al. 2012). Together, these studies highlight the need to develop models that can faithfully replicate aspects of disseminated and fatal RMSF in mammals so that putative therapeutics may more accurately be tested.

Several animal models have been developed to study the initiation of rickettsial infection and the progression to disease (Eisemann and Osterman 1976; Walker et al. 1994; Ellison et al. 2008). Interestingly, the C3H/HeN mouse strain was utilized to demonstrate that sub-lethal inoculation of R. australis elicited complete protective immunity when subsequently challenge with lethal doses of the related pathogen, R. conorii (Feng and Walker 2003). The results suggested that nearly identical antigens from related rickettsial species were sufficient to elicit broad-spectrum protective immunity. Indeed, previous experiments demonstrated that the widely conserved rickettsial surface cell antigen, Sca5/OmpB is a highly immunogenic rickettsial antigen with potential protective attributes against SFG rickettsial diseases (Anacker et al. 1985; Li, Lenz and Walker 1988; Feng et al. 2004). We recently demonstrated that active vaccination of animals with recombinant R. conorii OmpB passenger domain (aa 34–1338) is sufficient to elicit protective immunity against fatal outcomes in a C3H/HeN model of MSF (Chan et al. 2011). Other studies also evaluated the efficacy using a portion of OmpB to generate protective immunity against R. rickettsii infections (Gong et al. 2014, 2015). The OmpB passenger domains from closely and distantly related rickettsial species exhibit a great deal of amino acid identity and similarity (Blanc et al. 2005) suggesting that OmpB proteins may elicit cross-protective immunity against a variety of rickettsial species (Feng and Walker 2003).

Herein, we confirm that the C3H/HeN mouse strain is susceptible to infection with R. rickettsii strain ‘Sheila Smith’ via intravenous inoculation and that immunization with R. rickettsii OmpB protects these animals from fatal disease. Furthermore, we demonstrate that vaccination of animals with R. conorii OmpB can stimulate antibodies that recognize native OmpB at the surface of R. rickettsii, but that the immune response is not sufficient to elicit protective immunity against the related pathogen, R. rickettsii.

MATERIALS AND METHODS

Cell lines and bacterial strains

African green monkey kidney cells (Vero, ATCC CCL-81 cells) were cultured and maintained under standard conditions as described previously (Chan et al. 2009). Escherichia coli BL21 (DE3) or TOP10 were grown in LB Miller broth at 37°C supplemented with carbenicillin (50 μg ml⁻¹) or kanamycin sulfate (50 μg ml⁻¹) where needed. Rickettsia rickettsii strain ‘Sheila Smith’ was propagated and isolated from Vero cell cultures similarly as described (Chan et al. 2011). Rickettsia rickettsii were purified from Vero cells by needle lysis and centrifugation over a 20% sucrose cushion (16 000 × g, 4°C, 30 min) and stored at −80°C in SPG buffer (218 mM sucrose, 3.8 mM KH₂PO₄, 7.2 mM K₂HPO₄, 4.9 mM L-glutamate, pH 7.2).

Quantification of R. rickettsii infectious titers

Enumeration of viable and infectious R. rickettsii was determined by limiting dilution and infection of Vero cells as described in (Chan et al. 2011). Rickettsia rickettsii-positive wells were counted and the infectious titer, presented as infectious units ml⁻¹, was calculated.

Plasmid construction

Plasmids pYC82 and pEC3, containing R. conorii sca1_5264-5479 (GenBank AAL02557.1) and murine actin, respectively, and plasmid pYC69 harboring His₆-SUMO-Rc ompB_35-1334 have been previously described (Chan et al. 2011). Rickettsia rickettsii rompB (base pairs 103–4002), encoding the passenger domain, was PCR amplified from R. rickettsia Sheila Smith chromosomal DNA using the forward primer 5^′-nnGGATCCGCTGCTATACAGCAGAATAG-3^′ and reverse primer 5^′-nnCTCGAGTTATAATCTGTTACCAAGTTGAGC-3and directionally cloned into pET28-Smt3 into the BamHI and XhoI sites to generate pYC90. The BamHI and XhoI restriction sites in the primer sequences are highlighted in bold.

Protein alignment

Amino acid alignments of the OmpB ‘passenger domain’ (comprised of R. conorii amino acids 35–1334) from Rickettsia spp. were performed using the ClustalW function in the MacVector software (MacVector, Cary, NC) using the input accession sequences Q9KKA3.2 (R. conorii), and Q53047.1 (R. rickettsii). The percent identity between the two sequences was automatically calculated using the MacVector software.

Protein expression and purification

Overnight bacterial cultures were diluted 1:20 into 1 L of fresh medium and grown at 37°C to mid-exponential phase (OD₆₀₀ = 0.5–0.6) and induced at the specified conditions. Cultures were cooled on ice for 30 min, and then induced using 0.1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25°C overnight. His-tagged rOmpB fusions (encoded by pYC69 and pYC90) were expressed and purified under native conditions from E. coli BL21 (DE3) (Stratagene). Bacteria were harvested, washed in phosphate buffered saline (PBS), lysed by passage through the French pressure cell 2X (10 300 kpa), and rOmpB fusions purified on the appropriate 5 ml HisTrap-FF column (GE Healthcare) using an ÄKTA FPLC (GE Healthcare). Fractions containing fusion proteins were pooled and dialyzed into TBS (50 mM Tris, 150 mM NaCl, pH 8), then snap-frozen in liquid nitrogen and stored at −80°C.

His₆-SUMO-RcOmpB_35-1334 (encoded by pYC69) and His₆-SUMO-RrOmpB_35-1333 (encoded by pYC90) used for immunizations were further processed to remove the affinity tag as described in Chan et al. (2011). Briefly, proteins in TBS containing 0.2% NP-40 and 1 mM DTT were cleaved at 4°C overnight with gentle rocking with SUMO protease 1 (0.01 U μg⁻¹ protein) (Life Sensors). SUMO protease (His₆-tagged) and liberated His₆-SUMO tag were separated from the untagged OmpB by affinity purification over Ni-nitrilotriacetic acid (Ni-NTA) agarose matrices (Qiagen). The flow-through containing untagged RcOmpB_35-1334 and RrOmpB_34-1333 was buffer-exchanged into PBS and stored at −80°C until used for mouse immunization studies. Protein concentrations were quantified by bicinchoninic acid assay (BCA) (Pierce).

Mouse challenges and immunizations

For active immunizations, groups (n = 15) of 5- to 7-week-old male C3H/HeN mice (Harlan Sprague Dawley) were immunized by intramuscular injection into the hind leg with 0.1 ml aliquots of 50 μg of recombinant RcOmpB and RrOmpB in PBS emulsified 1:1 with complete Freund's adjuvant (CFA) or PBS emulsified 1:1 with CFA as a negative control. Booster immunizations with proteins emulsified in incomplete Freund's adjuvant (IFA) or PBS emulsified in IFA as a control were done 21 and 42 days after initial immunization. Pre-designated animals from each group (n = 4 for PBS-immunized mice; n = 3 each for R. conorii- and R. rickettsii-OmpB vaccinated mice) were sacrificed and blood harvested via cardiac puncture on day 56 to determine serum antibody titers by ELISA against the immunization antigen (titer ± SEM).

Mice were anesthetized on day 63 with 100–130 mg kg⁻¹ ketamine and 3–6 mg kg⁻¹ xylazine and challenged by i.v. retro-orbital injection with 4 LD₅₀ R. rickettsii diluted in 0.1 ml SPG buffer (1LD₅₀ = 1.0 × 10⁷ infectious units). Infected mice were monitored at least twice daily for signs of disease and daily for weight loss. Animals that exhibited all of the following symptoms (ruffled fur, hunched posture, shallow respiration, immobility when touched and weight loss of >15% of the initial body weight) were removed from the study and scored as succumbing to the infection. On days 1 and 5 post-infection, mice from each experimental group were sacrificed and their spleens and livers aseptically extracted to determine R. rickettsii load by quantitative PCR as described below. Samples of the liver and spleen from these animals were also fixed in formalin and further processed for histology and immunohistochemistry as described below. Survival data were determined from 10 mice per group and repeated with similar results. Animal experiments were performed in accordance with institutional guidelines and protocols approved by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee (IACUC) at The University of Chicago and at the Louisiana State University School of Veterinary Medicine.

Enzyme-linked immunosorbent assay

Briefly, 96-well MaxiSorp plates (Nunc) were coated with either 1 μg of His₆-SUMO-RrOmpB_35-1333 or 1 μg of His₆-SUMO-RcOmpB_35-1333 in bicarbonate buffer (0.1 M Na₂CO₃, 0.1 M NaHCO₃, pH9.6) overnight at 4°C. Murine sera at the indicated concentrations were incubated in triplicate wells, and subsequently developed with the appropriate rabbit anti-mouse HRP-conjugated (1:10 000) secondary antibodies and a TMB substrate kit (Pierce). Colorimetric differences were analyzed with a spectrophotometer at 450 nm. Serum titers against homologous antigens are presented as (titer ± SEM): PBS (<400, below the limit of detection); R. conorii rOmpB (110 670 ± 18 706); R. rickettsii rOmpB (126 107 ± 16 171).

Flow cytometry

Vero cell-purified R. rickettsii were fixed for 20 min in 4% paraformaldehyde (PFA) in PBS and subsequently washed in cold PBS. Immune serum recognition of R. rickettsii was determined by flow cytometry using a FACSCanto (BD Biosciences, San Jose, CA) with forward scatter, side scatter and 488 nm excitation and 530/535 nm detection. Deposition of IgG from each mouse serum sample (1:200) was detected using AlexaFluor488 goat anti-mouse IgG antibody (Life Technologies, Grand Island, NY), and fluorescence values were analyzed using the FlowJo software (Tree Star, Ashland, OR).

PCR quantification of R. rickettsii levels

R. rickettsii loads in the spleen were quantified by TaqMan quantitative PCR (qPCR). Sections of spleen were harvested from two mice from each group sacrificed one (1) day post-infection and stored at −80°C until processing. Total chromosomal DNA from digested tissue homogenates was purified as described (Chan et al. 2011), eluted in 50 μl of H₂O and normalized to 50 μg ml⁻¹. TaqMan qPCR reactions were run in multiplex, with primer sets used to construct pEC3 (R. rickettsii sca1) and pYC82 (M. musculus actin) and fluorescent probes (Integrated DNA Technologies) annealing to sca1 and actin in each reaction well and analyzed on the LightCycler 480 II system (Roche). Standard curves were generated using pEC3 and pYC82. PCR reactions were run as follows: 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 58°C for 60 s. VIC (4,7,2^′-trichloro-7-phenyl-6-carboxyfluorescein) and FAM (6-carboxyfluorescein) fluorescence emissions were recorded for each reaction at the 58°C stage. Loads of R. rickettsii in organs were expressed as the average number of sca1 copies per murine actin gene copies.

Indirect immunohistochemistry

Murine tissues, including liver and spleen, were collected immediately after euthanasia, fixed in 10% buffered formalin, and then further processed for histology. For indirect immunohistochemistry, slides were deparaffinized in three changes of xylene for five minutes each. The samples were then rehydrated in three changes of 100% alcohol and one change of 95% alcohol for 5 min each before being rinsed three times in deionized water. Antigen retrieval was performed with 10mM citrate buffer (pH 6.0) for 10 minutes at 125–127°C (Biocare Decloaker) and then slides were cooled for 20 min before being rinsed three times each with deionized water first and Tris-buffer with 1% Tween-20 (pH 7.6). The rest of the procedure was performed in a DAKO AutoStainer LINK 48. Endogenous peroxide activity was blocked with 3% hydrogen peroxide for 3 min before a buffer rinse. Nonspecific antibody binding was blocked by incubation with normal goat serum for 30 min (Vector S1000, Vector Laboratories, Burlingame, CA) followed by incubation with no primary antibody as a control or rabbit anti-RcPFA (1:2000 dilution) that recognizes R. rickettsii and R. conorii (Chan et al. 2011) for 30 min followed by a buffer rinse. The sample was then incubated with a 1:1000 dilution of biotinylated anti-rabbit IgG secondary antibody (Vector BA 1000, Vector Laboratories, Burlingame, CA) for 30 min followed by a buffer rinse. The tissue samples were then exposed to the detection reagent avidin/biotinylated enzyme complex (Vectastain ABC Rabbit IgG Kit, Vector PK 6102, Vector Laboratories, Burlingame, CA) for 30 minutes before a buffer rinse and application of NovaRed (Vector Sk-4800, Vector Laboratories, Burlingame, CA) followed by a buffer rinse and then a rinse with deionized water. The samples were counterstained for 5 min with Mayers hematoxylin (Lillie's modification) (Dako, North America, Inc, Carpinteria, CA) rinsed again with buffer and then deionized water, and removed from the stainer. Slides were dried in an oven at 60°C for 30 min before being removed and cooled for 5–10 min. Slides were rinsed in three changes of xylene for three minutes each before being coverslipped. Slides were analyzed and pathologic changes recorded by F. Del Piero, DVM, Ph.D., Diplomate of the American College of Veterinary Pathologists (DACVP).

Statistical analyses

Statistics were computed in GraphPad Prism using the Log-rank (Mantel-Cox) Test function. In studies pertaining to the active immunizations, OmpB-immunized mouse groups (RcOmpB and RrOmpB) were compared against PBS-group values (PBS vs. RcOmpB; PBS vs. RrOmpB) and to each other (RcOmpB vs. RrOmpB). All statistically significant comparisons, where P < 0.05, are reported.

RESULTS

Induction of high titer antibodies responses against recombinant OmpB proteins in C3H/HeN mice

OmpB proteins from R. rickettsii and R. conorii exhibit 92% identity at the amino acid level (Fig. 1). To address the potential cross-protective contribution of immune responses generated against a conserved rickettsial antigen, OmpB, we expressed and purified recombinant versions of the OmpB passenger domain from both R. conorii and R. rickettsii (Fig. 2A) and then independently immunized mice with these constructs. Cohorts of mice were mock immunized with PBS to serve as controls. A small subset of mice was sacrificed, blood harvested and humoral immune responses against OmpB were determined by ELISA (n = 4 for PBS-immunized mice; n = 3 each for R. conorii- and R. rickettsii-OmpB vaccinated mice). Immunization with R. conorii and R. rickettsii OmpB passenger domain elicited strong antibody responses against the homolgous purified recombinant protein. Mice immunized with PBS did not exhibit any significant humoral immune reactivity towards OmpB (Fig. 2B).

Figure 1.

Amino acid alignment of the OmpB passenger domain from related rickettsial species. OmpB proteins from R. rickettsii strain ‘Sheila Smith’ and R. conorii strain Malish 7 exhibit a significant amount of identity and similarity. Identical amino acids are boxed and shaded in grey. A generated consensus sequence is listed below the two query sequences.

Figure 2.

Both R. rickettsii and R. conorii OmpB passenger domains elicit strong immune IgG responses against R. rickettsii. (A) Recombinant OmpB proteins from R. conorii (RcOmpB) and R. rickettsii (RrOmpB) were expressed and purified under native conditions from BL21 (DE3) cells harboring pYC69 and pYC90, respectively. Representative Coomassie stained SDS-PAGE gels of purified proteins are shown. Lane 1, His₆-SUMO tagged OmpB; lane 2, SUMO protease treated samples; lane 3, dialyzed protein used for immunization. (B) Antibody titers from immunized mice. ELISA values are presented as titers +/− the standard error of the mean (SEM). Immunizations with control PBS did not generate any reactivity against either RrOmpB or RcOmpB (<400 represents values below the limit of detection). (C) Flow cytometric analyses of R. rickettsii recognition by immunized mouse sera. Sera from PBS-, RcOmpB- and RrOmpB-immunized C3H/HeN mice were analyzed by flow cytometry for IgG antibodies that could recognize OmpB epitopes on the R. rickettsii surface. Sera from four PBS- or three OmpB-vaccinated mice per group were analyzed. The intensity of fluorescence for each mouse is displayed as a different color (PBS, black; RcOmpB, red; RrOmpB, green). At least 100 000 events were measured per histogram.

Vaccination of mice with recombinant R. ricketsii OmpB, but not R. conorii OmpB, elicits protective immunity

We subsequently investigated whether the immune responses generated against recombinant OmpB proteins were sufficient to generate antibodies that recognize OmpB on the surface of purified R. rickettsii. Sera isolated from these mice were incubated with R. rickettsii and then serum recognition of surface associated OmpB was determined by flow cytometry. Sera from both RcOmpB- and RrOmpB-vaccinated animals contain antibodies that recognize OmpB on the surface of R. rickettsii (Fig. 2C). This finding lead us to hypothesize that vaccination with either recombinant protein will provide protection against a R. rickettsii challenge.

To assess the validity of this hypothesis, we challenged groups of 10 mice with ∼4 LD₅₀ of R. rickettsii, and monitored the animals for survival (Fig. 3A). Animals mock-immunized with PBS succumbed to the infection by day 5 with two animals surviving until day 8 and day 10. Several animals vaccinated with R. conorii OmpB (RcOmpB) exhibited signs of disease and began to succumb to infection similarly to the PBS vaccinated control group. In contrast, all animals vaccinated with R. rickettsii OmpB (RrOmpB) remained otherwise healthy with no overt clinical signs of disease during the 21-day time course of the experiment. In addition, the levels of R. rickettsii present in mice immunized with recombinant RrOmpB protein were quantitatively lower in the spleen compared to the PBS- and RcOmpB-vaccinated animals (Fig. 3B). On day 5 post-infection, the spleens of infected PBS- and RcOmpB- vaccinated mice exhibited severe lymphoid follicle hyperplasia, perifollicular moderate neutrophilic splenitis and bacteria in tingible body macrophages (Fig. S1A—F, Supporting Information). The liver of RcOmpB- and PBS vaccinated-animals exhibited severe, multifocal, random, necrotizing and neutrophilic hepatitis (Fig. 4A and C) with bacteria within Kupffer cells, hepatocytes, neutrophils and some lymphocytes (Fig. 4B and D). Bacterial colonization was reduced in both the spleen and liver in R. rickettsii OmpB vaccinated animals compared to the PBS-vaccinated and RcOmpB-vaccinated mice (Figs S1F and S4F, Supporting Information). These results demonstrate that immunization with recombinant R. rickettsii OmpB but not with R. conorii OmpB is efficacious in eliciting protective immunity against fatal R. rickettsii infection.

Figure 3.

Immunization with recombinant R. rickettsii but not R. conorii OmpB triggers protective immunity against fatal R. rickettsii infections. Cohorts of animals were immunized with PBS (control) and the indicated OmpB proteins and then challenged with 4LD₅₀ of R. rickettsii ‘Sheila Smith’. (A) Vaccination with R. rickettsii OmpB, but not PBS or R. conorii OmpB prevents fatal outcomes in C3H/HeN mice. (PBS vs. RcOmpB P = 0.1622; PBS vs. RrOmpB P < 0.0001; RcOmpB vs. RrOmpB P = 0.0009). (B) Rickettsia rickettsii colonization in the spleen at day 1 post-infection. Immunization with R. rickettsii OmpB (RrOmpB) diminishes colonization as determined by qPCR. Data are presented as the ratio of copies of R. rickettsii sca1 vs. copies of murine actin from two mice per experimental group. The presence of R. rickettsii in the RrOmpB vaccinated animals was below the level of detection (not detectable, n.d.).

Figure 4.

Fatal outcomes are associated with severe hepatic lesions in R. rickettsii challenged animals. Left-hand column hematoxylin and eosin stain. Right-hand column: anti-R. rickettsii immunohistochemistry (brown) with hematoxoxylin stain. (A) and (B) PBS-vaccinated animals: multifocal to coalescing coagulative hepatic necrosis characterized by the presence of numerous neutrophils and leukocytic debris. IHC staining of this lesion revealed the presence of numerous bacteria within the cytoplasm of macrophages and neutrophils. (C) and (D) RcOmpB-vaccinated animals: large pyogranuloma with necrosis. Focal necrosis is prominent with neutrophils and macrophages containing numerous intracytoplasmic bacteria. (E) and (F) RrOmpB-vaccinated animals: moderate neutrophilic and pyogranulomatous hepatitis. IHC staining revealed rickettsial antigen within Kupffer cells and other macrophages and in small pyogranulomas.

DISCUSSION

Misdiagnosis of rickettsial diseases can often lead to a delay in the administration of effective antibiotics and can result in severe morbidity and mortality (Walker 1989a,b). In addition, a vaccine against RMSF is not currently available, necessitating the development of novel therapies against these infections. We have previously demonstrated that OmpB from R. conorii is sufficient to mediate protective immunity against R. conorii infections in C3H/HeN mice (Chan et al. 2011). We and others (Gong et al. 2015) have now demonstrated that vaccination of C3H/HeN mice with recombinant OmpB protein derived from R. rickettsii generated specific high titer antibody responses and afforded protective immunity against fatal RMSF. Sera isolated from these vaccinated animals contained antibodies that recognized OmpB at the outer-membrane of intact R. rickettsii cells suggesting that at least some portion of the generated antibodies were neutralizing. RrOmpB-vaccinated mice exhibited minimal R. rickettsii colonization in the liver and spleen throughout the time course of the infection and were protected from fatal outcomes.

Interestingly, vaccination of animals with R. conorii OmpB (RcOmpB) also elicited high titer antibodies that were capable of recognizing OmpB on the surface of R. rickettsii. However, this immunization scheme failed to elicit strong efficacious protective immunity. The reason for this difference is unclear; however, it is possible that the slight differences in amino acid sequences between RcOmpB and RrOmpB may be enough to account for the failure to elicit the proper immune responses (Fig. 1). Alternatively, minor sequence differences between the two OmpB proteins may result in conformational changes in protein structure such that neutralizing antibodies generated against one antigen may not recognize a heterologous antigen and thus may not be protective. We have previously demonstrated in a murine model of MSF that generation of specific R. conorii OmpB high titer antibodies that recognize OmpB on the surface of R. conorii is alone not a correlate of protective immunity (Chan et al. 2011). Neutralizing antibodies that afforded protective immunity were likely directed against conformation specific epitopes on OmpB as has been previously suggested (Anacker et al. 1985) and importantly were capable of fixing complement (Chan et al. 2011). This hypothesis was further supported by the demonstration that non-protective OmpB monoclonal antibodies react with OmpB at the surface of intact bacteria and map to a C-terminal domain (amino acids 693–1334) on a denatured recombinant OmpB protein, while protective monoclonal antibodies could not be mapped to any region of a denatured OmpB protein (Chan et al. 2011). Direct analysis of the conformational epitopes on OmpB required to generate protective antibody responses has been hindered by the lack of structural information for this and other rickettsial surface antigens. Furthermore, we have demonstrated that immunization of animals with RcOmpB is sufficient to generate antibodies that cross-react against OmpB on the surface of R. rickettsii (Fig. 2), but are not protective. Thus, the use of a single protein-based vaccine may not be sufficient to elicit broad-spectrum protective immunity against other related rickettsial species. The mechanism of protective immunity mediated by vaccination with RrOmpB in the C3H/HeN model of RMSF warrants further investigation. In light of the increase in the frequency and morbidity and mortality of R. rickettsii infections in humans, development of active and efficacious immune therapies is of critical importance.

SUPPLEMENTARY DATA

Supplementary data are available at FEMSPD online.

FUNDING

This work was supported by an award by the National Institute of Allergy and Infectious Diseases (NIAID), Infectious Diseases Branch [AI 72606 to JJM]. The authors also acknowledge support from the Region V Great Lakes Regional Center of Excellence (GLRCE) in Biodefense and Emerging Infectious Diseases Consortium [GLRCE, U54-AI 057153]. This work was also supported in part by the Genetics and Regulation Training Grant [T32 GM007197 to Y.G.Y. Chan] and in part by the Molecular Cell Biology Training Grant [T32 GM007183 to MMC] at the University of Chicago.

Conflict of interest. None declared.