The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Awasthi, S.
Right arrow Articles by Coalson, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Awasthi, S.
Right arrow Articles by Coalson, J. J.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Immunization
The Journal of Immunology, 2005, 175: 3900-3906.
Copyright © 2005 by The American Association of Immunologists

Efficacy of Antigen 2/Proline-Rich Antigen cDNA-Transfected Dendritic Cells in Immunization of Mice against Coccidioides posadasii1,2

Shanjana Awasthi3,*, Vibhudutta Awasthi{dagger}, D. Mitchell Magee4,{ddagger} and Jacqueline J. Coalson*

* Department of Pathology, {dagger} Department of Radiology, and {ddagger} Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Coccidioides posadasii causes coccidioidomycosis, or Valley fever, in the endemic regions of the Southwestern United States. The susceptibility to C. posadasii infection has been attributed to a decreased Th1 cellular response. APCs, especially dendritic cells (DCs), play an important role in the activation of Th1 response. In this study, we investigated the efficacy of a DC-based vaccine against C. posadasii in a mouse model of coccidioidomycosis. We intranasally immunized C57BL6 mice with syngeneic, bone marrow-derived DCs (JAWS II cells) transfected with a cDNA encoding the protective Coccidioides-Ag2/proline-rich Ag. The immunized mice were lethally challenged with C. posadasii through either an i.p. or intranasal route. Upon necropsy after 10 days of infection, fungal burden in lung and spleen of immunized mice was significantly reduced as compared with the control animals. The lung tissue homogenates of immunized animals showed higher levels of IFN-{gamma}. Histologically, lung tissues of immunized mice were in better condition than the control mice. To further investigate, we studied the biodistribution and trafficking of injected DCs by nuclear imaging techniques. For this purpose, the transfected DCs were radiolabeled with 111In-oxime. Scintigraphic images showed that most of the label remained in the gastrointestinal tract. A significant amount was also observed in lung, but there were negligible circulating 111In label in blood. The results suggest that the DCs have a potent immunostimulatory activity, and immunization with DCs transfected with Ag2/proline-rich Ag-cDNA induces protective immunity against C. posadasii in C57BL6 mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Coccidioides is endemic in the Southwestern regions of the United States, and parts of Mexico, Central, and South America (1). It has been divided into two species: immitis for the isolates from California and posadasii for all the other isolates (2). The fungus propagates in soil in the form of mycelia that produce arthroconidia (4–6 µm). Inhalation of aerosolized arthroconidia is the primary route of infection. Once inside the host, the arthroconidia develop into endosporulating spherules that gradually spread systemically. The clinical manifestations of the disease range from a primary benign pulmonary infection to a progressive pulmonary or extrapulmonary disease involving skin, bones, CNS, and other organ systems (3). On the basis of a wide recognition of its potential use in bioterrorism, Coccidioides has been included in the list of Select Agents (4).

Due to the virulent nature of Coccidioides, endemicity of infection and frequent relapse after chemotherapy, an urgent need for the development of an effective therapy against coccidioidomycosis has been acknowledged (1, 5). It has been established that the protective immunity against Coccidioides posadasii is mounted by a Th1 cell response (6, 7). Among different mouse strains, BALB/c and C57BL6 mice are susceptible to Coccidioides, whereas DBA/2 mice are resistant to Coccidioides infection (8). The genetic basis of this difference is not clear, but it appears to be associated with depressed Th1 cell reactivity (6, 9). For an efficient protective response, the Ags are required to be carried to the lymph nodes where subsequent activation of naive lymphocytes occurs. Dendritic cells (DCs)5 are the APCs, which are specialized in capturing the Ags and then migrating to the lymph nodes (10). These unique capabilities of DCs have been recently used to design immunotherapy against cancers and infectious diseases (11, 12, 13). The induction of immunity after immunization with DCs depends on the factors such as route of administration, cell-homing, Ag, and phenotype of DCs (10, 14).

A prerequisite to a successful DC-based vaccine is the identification of a potent Ag for in vitro stimulation of DCs. Specific to Coccidioides, different Ags have been tested in combination with a variety of adjuvants (15, 16, 17, 18). Among these, Ag2/proline-rich Ag (Ag2/PRA) has been identified as having a potent antigenicity (19, 20). In this work, we studied the immunostimulatory potential of syngeneic DCs that had been transfected with Ag2/PRA cDNA. We found that the immunization conferred protection in mice against Coccidioides infection. We further imaged trafficking of intranasal 111In-labeled DCs and found that the instilled DCs remained localized in the gastrointestinal tract (g.i.t.) for a prolonged period. The accumulation of radioactivity in lung was moderate but sustained.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The studies were approved by the Institutional Animal Care and Use Committee and Environmental and Health Safety Committee. C57BL6 mice (6 wk old; The Jackson Laboratory) were housed at the Laboratory Animal Resources in the University of Texas Health Science Center at San Antonio (UTHSCSA). They were provided food and water ad libitum. The mice that were infected with Coccidioides were housed in a biosafety level-3 facility. The animals receiving radiolabeled DCs were housed in isolation during the study.

Transfection of DCs with Ag2/PRA cDNA

An immortalized dendritic JAWS II cell line derived from bone marrow of C57BL6 mice was obtained from American Type Culture Collection. The Ag2/PRA cDNA was ligated to a pVR1012 vector containing VP22 insert from pVP22/myc-His (Invitrogen Life Technologies). Vector plasmid pVR1012 containing VP22 insert was used as a control. The cells were transiently transfected with 1.0 µg of DNA per 1 x 106 cells using TransIT-TKO (Mirus) (21). The transfected cells were maintained in complete {alpha}-MEM containing ribonucleosides, deoxyribonucleosides, 20% FBS, 4 mM L-glutamine, 5 ng/ml recombinant mouse GM-CSF, 100 µg/ml penicillin, 100 U/ml streptomycin, and 50 µg/ml gentamicin. After 24 h, the cells were collected and washed with low-endotoxin Dulbecco’s PBS (D-PBS). Viability of the transfected cells was assessed by trypan blue dye exclusion. Finally, the cells were counted and suspended in D-PBS (33–50 million per milliliter); 30 µl was used for intranasal instillation.

The expression of Ag2/PRA protein in transfected cells was confirmed by dot-immunoblot assay. Briefly, cell homogenates in a buffer containing protease inhibitors were loaded on a prewetted nitrocellulose membrane and blotted with anti-Ag2/PRA Ab; recombinant Ag2/PRA Ag was run as a control (21).

Flow cytometric analysis

The phenotype of transfected cells was studied by flow cytometric analysis after staining with fluorochrome-conjugated Abs against various cell surface markers. The cells (1 x 106 per 100 µl of D-PBS with 1% FBS) were incubated with 1 µg of rat anti-mouse CD16/CD32 Ab for 15 min. The cells were then stained with FITC-labeled rat anti-mouse CD14, PE-labeled hamster anti-mouse CD80, PE-labeled hamster anti-mouse CD11c, FITC-labeled rat anti-mouse CD86, biotin-conjugated rat anti-mouse CD40, or biotin-labeled rat anti-mouse MHC class II Abs (all from BD Pharmingen). After 30 min, the cells were incubated with streptavidin-allophycocyanin conjugate. Appropriate isotype-matched control Abs were used to determine the background level. The fluorescence analysis was performed using a FACSCalibur flow cytometer (BD Immunocytometry Systems). The histogram charts were analyzed using CellQuest version 3.1 software provided with the system.

C. posadasii

All experiments with C. posadasii were performed in a biosafety level-3 facility. C. posadasii strain Silveira was cultured on 1% glucose-0.5% yeast extract agar plates. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter) from the 6- to 8-wk-old mycelial-phase cultures. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements. They were enumerated on a hemocytometer and their viability was confirmed by plating on 1% glucose-0.5% yeast extract agar. The arthroconidia suspension was used for i.p. and intranasal injection in mice.

Mice immunization and C. posadasii infection

C57BL6 mice were anesthetized by i.m. injection of ketamine-xylazine (75 µg/g and 10 µg/g body weight, respectively). Transfected DCs (1–1.5 x 106 per 30 µl) were intranasally administered in both nares alternately. Comparative controls were Ag2/PRA plasmid DNA alone, nontransfected cells, and cells transfected with the vector plasmid alone. Mice were held in an upright position for 2 min to allow resumption of normal breathing. The immunization was performed twice at an interval of 1 wk (on days 0 and 7). The second immunization was given to boost the immune response. From our prior experience, immunizations at 1 wk apart are optimum in terms of efficacy and the duration of the study. The immunized mice were infected with C. posadasii either i.p. or intranasally. Intranasal challenge was given on day 19 with 30 arthroconidia per 30 µl of saline, whereas the i.p. challenge was given on day 14 with 2500 arthroconidia per 500 µl of saline. The infected mice become symptomatic around day 9–10 and start dying on day 11. Therefore, the mice were sacrificed on 10th day of infection, and their lungs and spleens were collected. A 10-fold dilution of the tissue homogenates in saline was inoculated on mycobiotic agar plates (Difco) and incubated at 33°C. The number of mycelial colonies were counted and normalized with Gram-weight of tissues.

Lung histology samples were also collected from the animals on the 10th day postinfection. A tissue piece was fixed in 10% formaldehyde. Sections (4 µm) were cut and stained with H&E stain. The histological slides were randomly studied in a blinded fashion and the pathological changes were graded as follows: grade 0–1, no inflammation or patchy areas of inflammation, no organisms; grade 2, small areas of bronchiolar and peribronchiolar inflammation with few organisms and no necrosis; grade 3, inflammation and necrosis with many organisms in bronchiolar and peribronchiolar regions.

Measurement of IFN-{gamma}

A portion of lung tissue was homogenized in a buffer (50 mg of tissue in 1 ml of buffer) containing 1% Igepal CA-630, 0.01% SDS, 1 mg/L leupeptin, 1 mM EDTA, 0.9 mg/L pepstatin, and 0.2 mM PMSF. The homogenates were filtered through 0.2-µm filters and the IFN-{gamma} was measured by ELISA (BD Pharmingen). Briefly, Immulon 2 plate wells (Dynatech Laboratories) were coated overnight at 4°C with 100 µl of purified anti-mouse IFN-{gamma} Ab (5 µg/ml; BD Pharmingen) diluted in 0.1 M NaHCO3, pH 8.2. The coated wells were washed with PBS containing 0.05% Tween 20 and blocked with PBS containing 3% BSA. The wells were incubated with the homogenate supernatants or standard solutions of recombinant mouse IFN-{gamma} (BD Pharmingen). After overnight incubation, biotinylated anti-mouse IFN-{gamma} Ab (100 µl, 2.5 µg/ml) was added. The wells were further incubated with streptavidin-peroxidase conjugate, followed by tetramethylbenzidine substrate solution. The reaction was stopped by adding 50 µl of 0.2 N sulfuric acid, and the plates were read at 450 nm. The limit of detection for IFN-{gamma} in this assay was 10 pg/ml.

Radiolabeling and trafficking of transfected DCs

To elucidate the distribution of intranasally administered DCs, the transfected cells were radiolabeled with a radiochemical 111In-oxime (Amersham). Briefly, the transfected DCs were gently scraped, twice washed, and suspended in ~1 ml of D-PBS. 111In-Oxime (1 mCi, 1 ml) was added drop-wise to the cell suspension with a gentle swirling. The labeling was allowed to occur for 30 min at room temperature. The radiolabeled cells were washed free of any unincorporated radioactivity by centrifugation (5 min, 500 x g) and washing twice with D-PBS. Radiolabeled pellet of cells was resuspended in D-PBS (33–50 million cells per milliliter).

A 30-µl aliquot of 111In-labeled DCs was administered in mice intranasally as described above. The animals were imaged using a small animal XPECT-CT system (Gamma Medica) at 6, 24, and 48 h after instillation. Both single photon emission computed tomograms (SPECT) as well as computed tomograms (CT) were acquired. During imaging, the animals were under isoflurane anesthesia (1% in 1 L/min oxygen). Anatomic localization of radioactivity was performed by fusing the SPECT images with the CT images using software provided with the system. For biodistribution studies, five mice each were sacrificed at 6, 24, 48, and 72 h after administration of radiolabeled DCs. Various organs were harvested, and the associated radioactivity was detected in an automated gamma counter (PerkinElmer). The percentage of radioactivity accumulation in various tissues was calculated on a per Gram tissue basis.

Statistical analysis

The results were analyzed by Student’s t test or Mann-Whitney U test using Prism software (GraphPad). The t test was used when the data were normally distributed; the rest were analyzed by Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characteristics of transfected DCs

DCs were transfected with Ag2/PRA cDNA with an efficiency of 30–50%, and the transfection was stable for at least 72 h (Fig. 1a). The cells expressed Ag2/PRA protein (Fig. 1b). Approximately 70% of transfected cells were viable at the time of immunization. Flow cytometry demonstrated a moderate increase in the expression of MHC class II, CD11c, CD40, CD80, and CD86 on the cell surface of transfected DCs as compared with nontransfected DCs (Fig. 2). The transfected cells were also radiolabeled with 111In-oxime to visualize in vivo trafficking of DCs. There was no change in the morphology and viability of cells after radiolabeling with 111In-oxime. Flow cytometry also showed no alteration in the fluorescence intensity values of positive-gated radiolabeled cells as compared with the nonlabeled ones (Fig. 2d).



View larger version (82K):
[in this window]
[in a new window]
  		FIGURE 1. a, Fluorescent micrograph of pHYG-EGFP- and Ag2/PRA cDNA-cotransfected DCs after 48 h of transfection. b, Expression of Ag2/PRA protein by Ag2/PRA cDNA-transfected DCs after 48 h of transfection. The intensity of the black dots is directly related to the amount of immunoreactive protein in the samples.

 


View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 2. Phenotype of nontransfected (NT-DC; a), vector plasmid DNA transfected (Vector-DC; b), Ag2/PRA cDNA-transfected (Ag2-DC; c), and unlabeled (U-DC; d) and 111In-labeled Ag2/PRA cDNA-transfected (In-DC) DCs. The mean fluorescent intensity values of cell surface markers in positive cells gated in M1 regions of histograms are in insets. Each set has an isotype control (Iso Con) for comparison.

 
Immunization of mice with Ag2/PRA cDNA-transfected DCs

The immunized mice were infected by live Coccidioides arthroconidia through either intranasal or i.p. administration (Table I). The fungal burden in lung and spleen was estimated by culturing the tissue homogenates. Based on the fungal colony counts, the mice immunized with Ag2/PRA cDNA-transfected DCs showed enhanced clearance of fungus as compared with the control mice immunized with vector plasmid-transfected DCs, nontransfected DCs, or Ag2/PRA cDNA alone (Table I; p < 0.001, p < 0.01, or p < 0.05, Mann-Whitney U test). Immunization with a lesser number of Ag2/PRA cDNA-transfected DCs did not induce reduction in fungal load (data not shown). The fungal colony counts were also lower in lung and spleen tissues of the mice immunized with Ag2/PRA cDNA alone. However, the relative fungal burden indicated that the protective effect of Ag2/PRA cDNA was enhanced when DCs were used as an adjuvant (Table I). Interestingly, despite the larger arthroconidia load with the i.p. infection compared with the intranasal infection (2500 vs 30), the reduction in fungal load was more pronounced in lungs of immunized mice challenged i.p. than in lungs of mice infected intranasally.


View this table:
[in this window]
[in a new window]
  		Table I. Fungal burden (104 cfu/g tissue, mean ± SEM) in immunized mice after intranasal (i.n.) and i.p. infection with C. posadasii arthroconidia

 
As an indication of lymphocyte activation, we also estimated IFN-{gamma} in the lung homogenate of animals receiving Ag2/PRA cDNA-transfected DCs or vector plasmid DNA-transfected DCs (Fig. 3). The amounts of IFN-{gamma} were found to be increased significantly in lung tissue homogenates from mice immunized with Ag2/PRA cDNA-transfected DCs as compared with the homogenates from mice immunized with vector plasmid DNA-transfected DCs (p < 0.05, t test). The increase was similar in both i.p. and intranasal infection. The lung histology indicated that the mice immunized with Ag2/PRA cDNA-transfected DCs presented with less tissue damage (grade 0–1 and 2), whereas tissue samples from mice immunized with vector plasmid DNA-transfected DCs showed grade 3 changes of multiple foci of necrosis, chronic inflammation, and many organisms (Figs. 4 and 5). Moreover, the lung tissue damage was significantly less when mice were infected i.p. (all with grade 1 injury) as compared with the intranasal injection (grade 1–3). Thus, the histology results corroborated with the observed reduction in fungal burden in immunized mice.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. IFN-{gamma} in lung tissue of immunized mice infected with C. posadasii arthroconidia. Data are shown as mean ± SEM of triplicate measurements from two separate experiments. *, p < 0.05 vs vector-transfected DCs.

 


View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 4. Histology grades in H&E-stained lung tissue sections from immunized mice infected with C. posadasii arthroconidia. Mice were immunized with either Ag2/PRA cDNA-transfected DCs (Ag2 DC) or vector plasmid-transfected DCs (Vector DC). **, p < 0.005 and *, p < 0.05 vs Vector DC.

 


View larger version (200K):
[in this window]
[in a new window]
  		FIGURE 5. Representative light microscopic photographs (x100) of H&E-stained slides of lung tissues from immunized mice infected with C. posadasii arthroconidia-Ag2 DC, i.p. infection (a); vector DC, i.p. infection (b); Ag2 DC, intranasal infection (c); and vector DC, intranasal infection (d).

 
Biodistribution and imaging

To investigate the in vivo trafficking of DCs after intranasal immunization, we radiolabeled transfected DCs with 111In-oxime. 111Indium is a gamma ray-emitting radionuclide that can be imaged using a gamma camera. The cell radiolabeling efficiency averaged ~60% and the viability of cells was not affected by the radiolabeling procedure. When the radiolabeled cells were cultured for up to 72 h under standard conditions, there was <20% loss of label in the media. This showed that the radiolabel was stably associated with the cells. The radiolabeled cells were administered intranasally in mice. The gamma camera images of animals showed pronounced accumulation of cells in the g.i.t. The accumulation in g.i.t. persisted for up to 48 h with negligible change in location (Fig. 6). There was a moderate accumulation of radioactivity in the lung while all the other organs exhibited negligible localization. An overlying CT image was simultaneously acquired in the same animal and was helpful in anatomically localizing the radioactivity in the animals. Similar observations were made in biodistribution experiments when the animals were sacrificed at 6, 24, 48, and 72 h after intranasal administration (Table II). Again, most of the radioactivity was seen associated with the upper g.i.t., i.e., esophagus and stomach. With time, g.i.t.-associated radioactivity rapidly decreased to background level. It appears that the g.i.t. radioactivity gradually cleared in the feces. No attempt was made to separate contents of the g.i.t. Lung, the other organ of major accumulation, showed relatively slower clearance of radiolabeled cells, and even after 72 h, ~23% of initial activity (at 6 h) was present in the lung tissue. Other tissues, including blood, did not show any accumulation of radiolabeled cells at any time point.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 6. A representative set of SPECT-CT mouse images administered with Ag2/PRA cDNA-transfected DCs labeled with 111In-oxime. The scintigraphic images are superimposed on CT images to enable anatomical marking of the radioactivity disposition.

 

View this table:
[in this window]
[in a new window]
  		Table II. Distribution of 111ln label in mice following intranasal instillation of 111ln-labeled DCs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Significant efforts have been made to develop a vaccine against Coccidioides during recent times. Various coccidioidal Ags have been identified as potential candidates including a spherule outer wall glycoprotein (22), a Coccidioides alkali-soluble, water-soluble extract (23) and {beta}-1,3-glucanosyltransferase (24). A killed spherule vaccine was shown to protect the mice and other animals from experimental Coccidioides infection, but failed to induce protection in humans in preclinical trials (25). The cell wall glycoprotein Ag2/PRA is the most extensively studied vaccine candidate against coccidioidomycosis. Different groups of investigators have found some success in the resolution of infection after immunization with Ag2/PRA using commercially available adjuvants (19, 20, 26, 27). Reports on efficacy of naked Ag2/PRA cDNA-based vaccine are inconsistent (19, 27, 28). Differences in route of immunization, route of challenge, numbers of arthroconidia, animal model, etc., make it difficult to compare the results from one study to another. It has been advocated that the genetic immunization and autologous DCs have many advantages over immunization with protein Ag and commercial adjuvants, respectively (29).

DCs are professional APCs capable of both priming a Th1-dependent immune reaction and efficiently stimulating memory response. Although present in small numbers, they are particularly localized in skin and mucosal surfaces (30). Thus, it makes sense to populate mucosal surfaces with Ag-pulsed DCs to confer Ag-specific immunity. DCs have been shown to phagocytose and present Ags from several pulmonary pathogens such as Mycobacterium tuberculosis (31) and fungal pathogens such as Aspergillus (32) and Candida albicans (33). Recently, introduction of conidia- or fungal-RNA-transfected DCs was found to induce protective response in vivo against C. albicans, Aspergillus fumigatus, and Cryptococcus neoformans (34, 35, 36). It has also been shown that peripheral blood-derived DCs, pulsed with a soluble extract of Coccidioides immitis spherules, can prime lymphocytes in vitro (37). Another ensuing study demonstrated that autologous, C. immitis Ag-pulsed DCs could reverse the in vitro anergic T cells response against C. immitis in patients with disseminated coccidioidomycosis (38). In Coccidioides infection, activated Th1 response is responsible for protective acquired immunity, and DCs may play an important role in connecting the innate immune response to the adaptive Th1 immunity (37, 38). These studies suggest that DC-based immune therapy could be useful in the treatment of disseminated coccidioidomycosis. Barring a few in vitro studies, no efforts have been made to study the potential of DCs for immunotherapy against C. posadasii infection in animal models.

Although viral transfection reagents are the mainstay in eukaryotic cell transfection, it has its own immunological drawbacks, and attempts are being made to replace them with novel nonviral reagents that can produce high transfection efficiency (39). We recently showed that TransIT-TKO reagent can be successfully used to transfect DCs with 30–50% transfection efficiency (21). Using the same technique, we transfected bone marrow-derived DCs and intranasally immunized mice to induce C. posadasii-specific immune response. Because lung is the first organ that encounters inhaled arthroconidia, our primary objective was to monitor lung pathology and microbial burden. We hypothesized that enhancing the C. posadasii-specific response in the lung may be beneficial against subsequent Coccidioides infection. Our results show that the immunization with Ag2/PRA cDNA-transfected DCs induces protective immunity and reduces fungal burden in C. posadasii-susceptible C57BL6 mice against a lethal challenge with C. posadasii. It appears that intranasal immunization confers immunity against infection from both pulmonary as well as i.p. route. A significant immunity was also observed when naked Ag2/PRA cDNA was administered, suggesting an important role of the components of mucosal barriers in immunological response against pulmonary infection. However, the protective effect is significantly enhanced when the immunization is performed with DCs as an adjuvant.

Quantitatively, compared with control mice, fungal burden in the lung tissue was reduced by a factor of 100 in immunized mice that were challenged i.p.; in the intranasal infection of immunized mice, the fungal reduction only occurred by a factor of 5 (Table I). The immunized mice also demonstrated better histological grading when they were i.p. infected as compared with the intranasal infection (Figs. 4 and 5). Perhaps a majority of nasally administered DCs travel to the gut, bypassing the trachea. To investigate this possibility, we radiolabeled transfected DCs with a gamma-emitting radionuclide 111In using an oxime complex. Apparently, radiolabeling does not alter the phenotypic properties of transfected DCs (Fig. 2). When intranasally administered, >65% label was found to accumulate in the g.i.t. comprising of esophagus, stomach, and intestine, while pulmonary accumulation was relatively moderate (11.4%). The radioactivity disappeared from the g.i.t. within 48 h of administration, but >23% of pulmonary radioactivity remained in the lung even after 72 h. The observation that the radioactivity was not seen in the systemic circulation, spleen, and other organs suggests that the intranasal DCs are capable of locally performing their function.

Migration of endogenous DCs is dependent on their interaction with the endothelial adhesion molecules and the chemokines produced at the local microenvironment (30). Once in the tissue, the DCs process the microbial Ags and attain maturity that is characterized by a down-regulation of chemokine receptors and an up-regulation of CCR7. The latter confers lymph node homing capability to the mature DCs (40). The current understanding of lymphatic homing of DCs is based on skin as a model. The mechanisms by which pulmonary or gut pathogens are monitored by the DCs are largely unknown (30). Although it has not been possible to demonstrate the direct involvement of pulmonary or gut lymphatics, the fact that the immunized mice showed protection against both i.p. and intranasal infection supports this conjecture. The scintigraphic images provide important visual impressions of the linear migration of cells in temporal fashion.

One drawback of this study is the use of bone marrow-derived DCs rather than the pulmonary DCs. Isolation of pulmonary DCs is difficult because they constitute <1% of total cells (41). Bone marrow is a rich source of DCs and has been used successfully for vaccine development and immunotherapy (11). In a clinical situation, bone marrow-derived DCs would be easier to obtain than the lung DCs. Therefore, we used well-characterized, immortalized C57BL6 mouse bone marrow-derived JAWS II cells. Another limitation of the study is the lack of any direct information on in vivo interaction of the DCs with naive lymphocytes leading up to the activation of the latter. Monitoring IFN-{gamma} in lung tissue partially mitigates this limitation. IFN-{gamma} is a major IFN produced by mitogenically or antigenically stimulated lymphocytes and considered as a direct indicator of lymphocyte activation (Fig. 3). Our findings that the immunization with Ag2/PRA cDNA-transfected DCs induces the protective immune response and reduces the fungal burden after lethal arthroconidia challenge, provides support for a role of DCs in the development of vaccine against C. posadasii.


   Acknowledgments
 
We thank Charles Thomas (FACS Facility, UTHSCSA) for FACS analysis. We also thank Adrian Donias, Amy Chein, Jamie Fritz, Anuradha Soundararajan, and Maxwell Amurao for their technical assistance. Dr. Rebecca Cox (UTHSCSA) provided Ag2/PRA Ag and Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by research grants from the California Health Care Foundation, the Department of Health Services of the State of California, California State University at Bakersfield, and the San Antonio Area Foundation from the Semp Russ Foundation. Back

2 Part of this work has been presented by S.A., D.M.M., and R.A. Cox at the Experimental Biology meeting held in Washington, D.C., April 17–21, 2004; by S.A. and V.A. at the Southwest Chapter Society of Nuclear Medicine meeting held in San Antonio, TX, March 14–16, 2005; and by V.A. and S.A. at the Society of Nuclear Medicine meeting held in Toronto, Canada, June 18–22, 2005. Back

3 Address correspondence and reprint requests to Dr. Shanjana Awasthi, Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: awasthis{at}uthscsa.edu Back

4 Current address: Arizona State University, Tempe, AZ 85287. Back

5 Abbreviations used in this paper: DC, dendritic cell; PRA, proline-rich Ag; CT, computed tomogram; D-PBS, Dulbecco’s PBS; g.i.t., gastrointestinal tract; SPECT, single photon emission computed tomogram. Back

Received for publication April 20, 2005. Accepted for publication July 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Galgiani, J. N.. 1993. Coccidioidomycosis. West J. Med. 159:153.-171. [Medline]
  2. Fisher, M., G. Koenig, T. White, J. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73.-84. [Abstract/Free Full Text]
  3. Mischel, P. S., H. V. Vinters. 1995. Coccidioidomycosis of the central nervous system: neuropathological and vasculopathic manifestations and clinical correlates. Clin. Infect. Dis. 20:400.-405. [Medline]
  4. Dixon, D. M.. 2001. Coccidioides immitis as a select agent of bioterrorism. J. Appl. Microbiol. 91:602.-605. [Medline]
  5. Pappagianis, D.. 2001. Seeking a vaccine against Coccidioides immitis and serologic studies: expectations and realities. Fungal Genet. Biol. 32:1.-9. [Medline]
  6. Ampel, N. M., L. Christian. 1997. In vitro modulation of proliferation and cytokine production by human peripheral blood mononuclear cells from subjects with various forms of coccidioidomycosis. Infect. Immun. 65:4483.-4487. [Abstract]
  7. Corry, D. B., N. M. Ampel, L. Christian, R. M. Locksley, J. N. Galgiani. 1996. Cytokine production by peripheral blood mononuclear cells in human coccidioidomycosis. J. Infect. Dis. 174:440.-443. [Medline]
  8. Kirkland, T. N., J. Fierer. 1983. Inbred mouse strains differ in resistance to lethal Coccidioides immitis infection. Infect. Immun. 40:912.-916. [Abstract/Free Full Text]
  9. Fish, D. G., N. M. Ampel, J. N. Galgiani, C. L. Dols, P. C. Kelly, C. H. Johnson, D. Pappagianis, J. E. Edwards, R. B. Wasserman, R. J. Clark, et al 1990. Coccidioidomycosis during human immunodeficiency virus infection: a review of 77 patients. Medicine 69:384.-391. [Medline]
  10. Steinman, R. M.. 2003. Some interfaces of dendritic cell biology. APMIS 111:675.-697. [Medline]
  11. Cerundolo, V., I. F. Hermans, M. Salio. 2004. Dendritic cells: a journey from laboratory to clinic. Nat. Immunol. 5:7.-10. [Medline]
  12. Fassnacht, U., A. Ackermann, P. Staeheli, J. Hausmann. 2004. Immunization with dendritic cells can break immunological ignorance toward a persisting virus in the central nervous system and induce partial protection against intracerebral viral challenge. J. Gen. Virol. 85:2379.-2387. [Abstract/Free Full Text]
  13. Shao, C., J. Qu, L. He, Y. Zhang, J. Wang, H. Zhou, Y. Wang, X. Liu. 2005. Dendritic cells transduced with an adenovirus vector encoding interleukin-12 are a potent vaccine for invasive pulmonary aspergillosis. Genes Immun. 6:103.-114. [Medline]
  14. Berger, T. G., E. S. Schultz. 2003. Dendritic cell-based immunotherapy. Curr. Top. Microbiol. Immunol. 276:163.-197. [Medline]
  15. Ampel, N. M.. 2003. Measurement of cellular immunity in human coccidioidomycosis. Mycopathologia 156:247.-262. [Medline]
  16. Barnato, A. E., G. D. Sanders, D. K. Owens. 2001. Cost-effectiveness of a potential vaccine for Coccidioides immitis. Emerg. Infect. Dis. 7:797.-806. [Medline]
  17. Cole, G. T., J. M. Xue, C. N. Okeke, E. J. Tarcha, V. Basrur, R. A. Schaller, R. A. Herr, J. J. Yu, C. Y. Hung. 2004. A vaccine against coccidioidomycosis is justified and attainable. Med. Mycol 42:189.-216. [Medline]
  18. Cox, R. A., D. M. Magee. 2004. Coccidioidomycosis: host response and vaccine development. Clin. Microbiol. Rev. 17:804.-839. [Abstract/Free Full Text]
  19. Jiang, C., D. M. Magee, T. N. Quitugua, R. A. Cox. 1999. Genetic vaccination against Coccidioides immitis: comparison of vaccine efficacy of recombinant antigen 2 and antigen 2 cDNA. Infect. Immun. 67:630.-635. [Abstract/Free Full Text]
  20. Shubitz, L., T. Peng, R. Perrill, J. Simons, K. Orsborn, J. N. Galgiani. 2002. Protection of mice against Coccidioides immitis intranasal infection by vaccination with recombinant antigen 2/PRA. Infect. Immun. 70:3287.-3289. [Abstract/Free Full Text]
  21. Awasthi, S., R. A. Cox. 2003. Transfection of murine dendritic cell line (JAWS II) by a nonviral transfection reagent. BioTechniques 35:600.-602, 604. [Medline]
  22. Hung, C. Y., N. M. Ampel, L. Christian, K. R. Seshan, G. T. Cole. 2000. A major cell surface antigen of Coccidioides immitis which elicits both humoral and cellular immune responses. Infect. Immun. 68:584.-593. [Abstract/Free Full Text]
  23. Ward, E. R., Jr, R. A. Cox, J. A. Schmitt, Jr, M. Huppert, S. H. Sun. 1975. Delayed-type hypersensitivity responses to a cell wall fraction of the mycelial phase of Coccidioides immitis. Infect. Immun. 12:1093.-1097. [Abstract/Free Full Text]
  24. Delgado, N., J. Xue, J. J. Yu, C. Y. Hung, G. T. Cole. 2003. A recombinant {beta}-1,3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis. Infect. Immun. 71:3010.-3019. [Abstract/Free Full Text]
  25. Pappagianis, D.. 1993. Evaluation of the protective efficacy of the killed Coccidioides immitis spherule vaccine in humans: The Valley Fever Vaccine Study Group. Am. Rev. Respir. Dis. 148:656.-660. [Medline]
  26. Kirkland, T. N., F. Finley, K. I. Orsborn, J. N. Galgiani. 1998. Evaluation of the proline-rich antigen of Coccidioides immitis as a vaccine candidate in mice. Infect. Immun. 66:3519.-3522. [Abstract/Free Full Text]
  27. Peng, T., L. Shubitz, J. Simons, R. Perrill, K. I. Orsborn, J. N. Galgiani. 2002. Localization within a proline-rich antigen (Ag2/PRA) of protective antigenicity against infection with Coccidioides immitis in mice. Infect. Immun. 70:3330.-3335. [Abstract/Free Full Text]
  28. Abuodeh, R. O., L. F. Shubitz, E. Siegel, S. Snyder, T. Peng, K. I. Orsborn, E. Brummer, D. A. Stevens, J. N. Galgiani. 1999. Resistance to Coccidioides immitis in mice after immunization with recombinant protein or a DNA vaccine of a proline-rich antigen. Infect. Immun. 67:2935.-2940. [Abstract/Free Full Text]
  29. Moll, H., C. Berberich. 2001. Dendritic cells as vectors for vaccination against infectious diseases. Int. J. Med. Microbiol. 291:323.-329. [Medline]
  30. Cavanagh, L. L., U. H. Von Andrian. 2002. Travellers in many guises: the origins and destinations of dendritic cells. Immunol. Cell Biol. 80:448.-462. [Medline]
  31. Gonzalez-Juarrero, M., I. M. Orme. 2001. Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis. Infect. Immun. 69:1127.-1133. [Abstract/Free Full Text]
  32. Bozza, S., R. Gaziano, A. Spreca, A. Bacci, C. Montagnoli, P. di Francesco, L. Romani. 2002. Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus. J. Immunol. 168:1362.-1371. [Abstract/Free Full Text]
  33. Romagnoli, G., R. Nisini, P. Chiani, S. Mariotti, R. Teloni, A. Cassone, A. Torosantucci. 2004. The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function. J. Leukocyte Biol. 75:117.-126. [Abstract/Free Full Text]
  34. Bacci, A., C. Montagnoli, K. Perruccio, S. Bozza, R. Gaziano, L. Pitzurra, A. Velardi, C. F. d’Ostiani, J. E. Cutler, L. Romani. 2002. Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation. J. Immunol. 168:2904.-2913. [Abstract/Free Full Text]
  35. Bozza, S., C. Montagnoli, R. Gaziano, G. Rossi, G. Nkwanyuo, S. Bellocchio, L. Romani. 2004. Dendritic cell-based vaccination against opportunistic fungi. Vaccine 22:857.-864. [Medline]
  36. Bozza, S., K. Perruccio, C. Montagnoli, R. Gaziano, S. Bellocchio, E. Burchielli, G. Nkwanyuo, L. Pitzurra, A. Velardi, L. Romani. 2003. A dendritic cell vaccine against invasive aspergillosis in allogeneic hematopoietic transplantation. Blood 102:3807.-3814. [Abstract/Free Full Text]
  37. Richards, J. O., N. M. Ampel, J. N. Galgiani, D. F. Lake. 2001. Dendritic cells pulsed with Coccidioides immitis lysate induce antigen-specific naive T cell activation. J. Infect. Dis. 184:1220.-1224. [Medline]
  38. Richards, J. O., N. M. Ampel, D. F. Lake. 2002. Reversal of coccidioidal anergy in vitro by dendritic cells from patients with disseminated coccidioidomycosis. J. Immunol. 169:2020.-2025. [Abstract/Free Full Text]
  39. Kovesdi, I., D. E. Brough, J. T. Bruder, T. J. Wickham. 1997. Adenoviral vectors for gene transfer. Curr. Opin. Biotechnol. 8:583.-589. [Medline]
  40. von Andrian, U. H., T. R. Mempel. 2003. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3:867.-878. [Medline]
  41. Masten, B. J., M. F. Lipscomb. 1997. Methods to isolate and study lung dendritic cells. C. Lenfant, Jr, and M. F. Lipscomb, Jr, and S. W. Russell, Jr, eds. In Lung Biology in Health and Disease: Lung Macrophages and Dendritic Cells in Health and Disease Vol. 102:223.-238. Marcel Dekker, New York.



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. McCormick, M. Santosuosso, C.-L. Small, C. R. Shaler, X. Zhang, M. Jeyanathan, J. Mu, S. Takenaka, P. Ngai, J. Gauldie, et al.
Mucosally Delivered Dendritic Cells Activate T Cells Independently of IL-12 and Endogenous APCs
J. Immunol., August 15, 2008; 181(4): 2356 - 2367.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
X. Jiang, C. Shen, J. Rey-Ladino, H. Yu, and R. C. Brunham
Characterization of Murine Dendritic Cell Line JAWS II and Primary Bone Marrow-Derived Dendritic Cells in Chlamydia muridarum Antigen Presentation and Induction of Protective Immunity
Infect. Immun., June 1, 2008; 76(6): 2392 - 2401.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
E. J. Tarcha, V. Basrur, C.-Y. Hung, M. J. Gardner, and G. T. Cole
Multivalent Recombinant Protein Vaccine against Coccidioidomycosis.
Infect. Immun., October 1, 2006; 74(10): 5802 - 5813.
[Abstract] [Full Text] [PDF]

Home page
 MicrobiologyHome page
R. D. Pathirana, N. M. O'Brien-Simpson, P. D. Veith, P. F. Riley, and E. C. Reynolds
Characterization of proteinase-adhesin complexes of Porphyromonas gingivalis.
Microbiology, August 1, 2006; 152(Pt 8): 2381 - 2394.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Awasthi, S.
Right arrow Articles by Coalson, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Awasthi, S.
Right arrow Articles by Coalson, J. J.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Immunization

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS