Immunization with a mycobacterial lipid vaccine improves pulmonary pathology in the guinea pig model of tuberculosis

Christopher C. Dascher1, Kenji Hiromatsu2, Xiaowei Xiong1, Caroline Morehouse1, Gerald Watts1, Gui Liu3, David N. McMurray4, Kenneth P. LeClair3, Steven A. Porcelli2 and Michael B. Brenner1

1 Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, and Harvard Medical School, Smith 552, 1 Jimmy Fund Way, Boston, MA 02115, USA 2 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Forchheimer 416, 1300 Morris Park Avenue, Bronx, NY 10461, USA 3 Antigenics, Inc., 34 Commerce Way, Woburn, MA 01801, USA 4 Department of Medical Microbiology and Immunology, College of Medicine, Texas A&M University System Health Science Center, College Station, TX 77843, USA

Correspondence to: C. C. Dascher; E-mail: cdascher{at}rics.bwh.harvard.edu
Transmitting editor: S. Koyasu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lipids and glycolipid molecules derived from Mycobacterium tuberculosis can be presented to T cells by CD1 cell-surface molecules in humans. These lipid-specific T cells are cytolytic, secrete pro-inflammatory cytokines and have bactericidal activity. Here, we describe studies in which lipids from M. tuberculosis were incorporated into liposomes with adjuvant and tested as vaccines in a guinea pig aerosol tuberculosis challenge model. Animals vaccinated with mycobacterial lipids showed reduced bacterial burdens in the lung and spleen at 4 weeks after infection. In addition, the lungs of lipid-vaccinated animals also had significantly less pathology, with granulomatous lesions being smaller and more lymphocytic. In contrast, animals receiving only vehicle control immunizations had granulomatous lesions that were larger and often contained caseous necrotic centers. Quantification of histopathology by morphometric analysis revealed that the overall percentage of lung occupied by diseased tissue was significantly smaller in lipid-vaccinated animals as compared to vehicle control animals. In addition, the mean area of individual granulomatous lesions was found to be significantly smaller in both lipid- and bacillus Calmette-Guerin-vaccinated guinea pigs. These data support an important role for lipid antigens in the immune response to M. tuberculosis infection, potentially through the generation of CD1-restricted T cells. Immunogenic lipids thus represent a novel class of antigens that might be included to enhance the protective effects of subunit vaccine formulations.

Keywords: bacterial infection, lung, rodent, vaccination


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Approximately 2 million deaths occur each year as a result of Mycobacterium tuberculosis infection, making it the world’s leading cause of mortality from a bacterial pathogen (1). Co-infection of M. tuberculosis, the causative agent of tuberculosis, with HIV has exacerbated the already overwhelming tuberculosis problem in many developing countries. Currently the only tuberculosis vaccine approved for human use is bacillus Calmette-Guerin (BCG), a live-attenuated strain of Mycobacterium bovis developed early in the 20th by Calmette and Guerin (2,3). Although effective in preventing disseminated tuberculosis in children, widespread use of the BCG vaccine in many countries has failed to significantly impact the spread of this disease in the adult population. Moreover, epidemiological studies of BCG vaccination programs have revealed a lack of consistency in its effects, with reported efficacy rates ranging from 0 to 80% (4). Therefore, there is an urgent need for improved vaccines against M. tuberculosis.

It is estimated that approximately a third of the world’s population has been infected with M. tuberculosis (1). While the number of people exposed is extremely high, only ~5% of infected individuals progress to active tuberculous disease. Therefore, an effective adaptive immune response is capable of controlling infection in the majority of infected individuals. Evidence derived from animal studies has emphasized the importance of T cells in the immune response to tuberculosis. These studies suggest that both MHC class I- and II-restricted T cells are required for effective control of M. tuberculosis infection (5,6). Thus, peptide antigens derived from bacterial proteins are important for the host immune response to M. tuberculosis infection.

In addition to the MHC, other antigen-presentation pathways may play a role in control and clearance of microbial infections. Our laboratory is investigating antigen presentation to T cells by the CD1 antigen-presentation pathway. In humans, the CD1 gene family consists of five non-polymorphic members designated CD1A, B, C, D and E. The CD1 proteins are cell-surface glycoproteins expressed by antigen-presenting cells including B cells and dendritic cells (7). Functional studies have established that CD1 has the unusual capacity to present bacterial lipids and glycolipids to T cells, thus expanding the possible targets available to the adaptive immune system for controlling infections. Interestingly, several lipid and glycolipid antigens that CD1 can present to T cells have been derived from M. tuberculosis and related mycobacterial species (813). These antigens include mycolic acid (8), lipoarabinomannan (9), glucose monomycolate (GMM) (10) and manosyl phophodolichol (11). The CD1-restricted T cells have important effector functions for controlling infections including cytolytic capacity, secretion of pro-inflammatory cytokines such as IFN-{gamma} and bactericidal activity (14,15). In addition, experiments demonstrate that patients recently infected with M. tuberculosis have increased CD1-restricted T cell responses to a lipid antigen (11). Taken together, these data indicate that CD1-restricted T cells may have a role in the cell-mediated immune response to tuberculosis.

The CD1 gene family can be divided into two groups based on sequence similarity, with group 1 consisting of the CD1a, b and c isoforms and the single CD1d isoform comprising group 2 CD1 (16). In addition, the group 1 and group 2 CD1 subsets may have different immune functions in the context of infections, with group 1 CD1 isoforms more relevant for presentation of foreign antigens and group 2 CD1 (CD1d) having a more immunoregulatory role (17). Importantly, all of the M. tuberculosis-derived lipid antigens described thus far are restricted to one of the group 1 CD1 isoforms. In contrast, no CD1d-restricted T cells that recognize lipid antigens derived from bacteria have been isolated. Thus, the choice of an animal model becomes critical for evaluating the role of group 1 CD1. Since mice have only the group 2 (CD1d) isoform, they are unsuitable for studies of group 1 CD1-restricted T cell responses (18). In contrast to mice and rats, we have shown that guinea pigs possess an extended family of group 1 CD1 genes including homologs of the human CD1 isoforms known to present mycobacterial lipids to T cells (19,20). In addition to having the group 1 CD1 homologs of interest for these studies, the guinea pig is a well-established small animal model for tuberculosis and has been extensively used in vaccine studies (2126). Hence, we chose the guinea pig model of tuberculosis for evaluating the efficacy of lipid antigens as tuberculosis vaccine components.

An effective subunit vaccine formulation should include a wide array of protective antigenic targets in order to stimulate different T cell subsets that possess distinct but complementary effector functions to control infection (27). The inclusion of lipid antigens that stimulate unique T cell subsets may provide additional effector functions that complement the MHC-restricted T cells. We have been investigating the use of the lipid antigen vaccination as a way of augmenting the protective T cell response to infection. We have shown previously that lipid antigen vaccination is capable of stimulating CD1-restricted T cell responses in guinea pigs (28). In the current study, we provide the first evidence that vaccination with mycobacterial lipid antigens is capable of reducing bacterial load and improving pathology following challenge infection with virulent M. tuberculosis that may correlate with protection.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Female Hartley strain guinea pigs (300 g) were obtained from Elm Hill Breeders (Chelmsford, MA). Uninfected guinea pigs were housed under specific pathogen-free conditions at the Dana Farber Cancer Institute Animal Resource Facility. Infected animals were housed in individual micro-isolator cages in a Biosafety Level 3 (BL3) animal facility. All appropriate animal use protocols were obtained and followed.

Bacteria
An agar plate of a spleen homogenate from a guinea pig infected by the aerosol route with M. tuberculosis strain H37Rv strain was kindly provided by Susan Phalen (Texas A & M, College Station, TX). A single colony of M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium supplemented with albumin–dextrose complex (ADC) and grown at 37°C until approximately mid-log phase. Aliquots were frozen at –80°C and titered to assure consistent viability. The BCG strain vaccine was used according to the manufacturer’s instructions (Statens Serum Institut, Copenhagen, Denmark).

Preparation and evaluation of lipid formulations
Whole lipid extracts where prepared from M. tuberculosis H37Rv grown in Middlebrook 7H9 (Difco, Detroit, MI) media made with glycerol or dextrose and supplemented with ADC (BBL, Cockeysville, MD) and 0.1% Tween 80 (w/v). Bacterial cell pellets were resuspended in ethanol and allowed to stand for 24 h. Ethanol was removed by evaporation under a nitrogen stream and the remaining cells were then lyophilized until completely desiccated. The non-covalently associated cell wall lipids were extracted from dried bacteria. Briefly, dried bacteria were mixed with chloroform:methanol (2:1), sonicated (Branson 1250; Branson Ultrasonics, Danbury, CT) and mixed overnight on a platform rocker. Insoluble material was removed by centrifugation and re-extracted with chloroform:methanol as above. The supernatants from the centrifugation steps containing soluble lipids were pooled and dried under a nitrogen gas stream, weighed and resuspended in chloroform:methanol (2:1), and stored at –20°C.

To evaluate possible protein contamination, samples of lipid extract were dried under nitrogen gas. An SDS–PAGE sample buffer was added and boiled for 5 min. A 15% polyacrylamide gel was run under reducing conditions as described (29). Bands were visualized using a silver-staining methodology as described (30). Samples of M. tuberculosis culture filtrate proteins and Ag85A (kindly provided by Dr John Belisle, Colorado State University, CO) were analyzed for comparison. Samples of lipid extracts were also analyzed for amino acid content as a marker of possible protein contamination by the Harvard University Microchemistry Facility. Briefly, a sample of lipid extract was subjected to acid hydrolysis, derivatized and analyzed by an ABI 420A amino acid analyzer (Applied Biosystems, Foster City, CA). Amino acid content was ~1% by weight according to this analysis. Approximately 50% of the amino acid content was made up of alanine and Glx (glutamine/glutamate).

Samples of lipid extracts were analyzed by thin-layer chromatography (TLC) as described previously (31). Known lipid antigens for CD1 were run on TLC as standards and included mycolic acid (M4537; Sigma-Aldrich, St Louis, MO) and Mycobacterium phlei GMM (kindly provided by D. B. Moody). Lipid standards for TLC were phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL). Samples for TLC were analyzed on a SilicaGel HPTLC glass-backed plate (Scientific Adsorbents, Atlanta, GA) using a chloroform:methanol:water solvent system (60:16:2) and visualized with cupric acid spray followed by heating. The lipid vaccine was formulated as liposome vesicles. Vehicle lipids were a 1:1 molar ratio of cholesterol:1,2-distearoyl-sn-glycero-3-phophocholine (DSPC) (Avanti Polar Lipids) mixed in chloroform. Two 15-ml borosilicate glass tubes (Kimble, Vineland, NJ) were designated as ‘vehicle’ and ‘vaccine’. Vehicle lipids (12 mg) were added to each tube. The vaccine tube received an additional 20 mg M. tuberculosis whole-lipid extract described above. Both tubes were then dried under a nitrogen gas stream to remove the volatile solvents (experiment 3). Alternatively, lipids were dried by a Rotovap (experiments 1 and 2). After drying, 1 ml adjuvant solution QS-21 (1 mg/ml in PBS; Antigenics, Woburn, MA) and/or 1 ml dimethyl-dioctadecylammonium bromide (DDA) (1 mg/ml in PBS; Eastman Kodak, Rochester, NY) were added along with PBS to yield a final volume of 3.5 ml for each tube. Both tubes were then sonicated for 30 min at 56°C in a water bath sonicator (Branson 1250). For experiment 3, the sonicated lipid mixture was then extruded with a mini-extruder (Avanti Polar Lipids) fitted with an 800-nm polycarbonate membrane according to the manufacturer’s instructions. Liposomes were transferred to sterile glass vials and sealed. Formulations were used the same day for immunization of animals. Each lipid vaccine dose contained 1 mg of M. tuberculosis lipids and 50 µg QS-21 or DDA or both (100 µg total) combined in a volume of 0.18 ml.

Immunization and challenge protocol
Animals were housed for 7 days prior to the start of vaccinations. All immunizations were s.c. injections on the flank using a single dose of liposome preparations. The vehicle control and lipid vaccine group was given a primary immunization followed by two booster immunizations at 3-week intervals. Following the last booster injection, animals remained in standard housing for 7 weeks. The positive control group received a single dose of BCG vaccine at the time of the final booster immunization of the lipid vaccine group. Following a 7-week rest period, guinea pigs were transferred to the BL3 animal facility and allowed to acclimate for 7 days prior to M. tuberculosis challenge. Purified protein derivative (PPD) skin tests were performed 1 week prior to transfer to BL3 housing using a 100 TU intradermal injection of PPD on the shaved flank of animals (Statens Serum Institut). Skin tests were measured at 48 h after PPD injection.

All 24 animals from a single vaccine trial were challenged simultaneously by aerosol exposure to M. tuberculosis H37Rv. For infection of animals, a single aliquot of M. tuberculosis H37Rv was thawed and diluted in 0.9% saline plus 0.1% Tween 80 (buffer ST), and added to a Lovelace nebulizer attached to a nose-only aerosol exposure chamber (Intox, Albuquerque, NM). Bacteria were diluted to a concentration predetermined by calibration experiments to deliver a dose of ~20 c.f.u. into the lungs of guinea pigs.

Animals were euthanized 4 weeks after infection by overdose of sodium pentobarbital. The spleen and right caudal lobe of the lung were sterilely removed and placed in separate Teflon-on-glass homogenizers containing 5 ml sterile ST buffer. Homogenized tissues were serially diluted and plated in duplicate on 7H11 agar plates (Remel, Lenexa, KS). The plates were incubated for 3 weeks at 37°C and then counted to determine the total c.f.u. in the lung lobe or spleen. The minimum detectable number of bacteria is ~50 c.f.u.

Electron Microscopy
A aliquot of 10 µl vehicle and lipid vaccine liposome preparations was removed just prior to injection into animals and subjected to electron microscopic analysis. Samples were dried onto degaussed copper grids and stained with 1% uranyl acetate. Samples were then visualized using a JEOL 100CX transmission electron microscope at 80 kV.

Tissue preparation and histology
Samples of all tissues from infected guinea pigs were fixed in 10% buffered Formalin for 7–14 days. Fixed tissues were embedded in paraffin, sectioned at 5 µm and stained with hematoxylin & eosin using standard techniques. All lung tissues were mounted in the same orientation and sectioned approximately through the same anatomical location.

Morphometric analysis of lung histopathology
Hematoxylin & eosin lung cross-sections were scanned at 4000 DPI with a Polaroid SprintScan slide scanner fitted with a PathScan Enabler (Meyer Instruments, Houston, TX). Digitized images were then analyzed using Scion (Frederick, MD) Image software in density slice mode with an LUD threshold range set at 40–200. The number of square pixels was calculated for each distinct lesion. The total diseased area (D) for the entire lung section was calculated by adding together the values for each lesion (lesions smaller than 500 pixels were excluded). The mean lesion cross-sectional area for each animal was calculated and the total area of the entire lung cross-section (T) was determined. The total percentage of diseased tissue was calculated by the formula D/T x 100. One random left caudal lobe cross-section from five animals was scanned per treatment group. Student’s t-test was used to determine statistical significance.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Kinetics of M. tuberculosis dissemination
The natural route of infection and transmission for M. tuberculosis in humans is by inhalation of bacteria contained in small (<10 µm) droplet nuclei expelled from the lungs by coughing. Therefore, we established a tuberculosis aerosol infection model for guinea pigs to approximate the natural mode of infection of this pathogen. The low-dose aerosol exposure system employed in these studies is similar to other models that have been described previously, with the exception that our system delivers bacteria in a nose-only mode instead of a whole-body exposure (32). Growth and dissemination of bacteria following infection of naive animals was examined in order to determine the typical load of organisms found in the lungs and spleen following aerosol infection. Figure 1 shows that growth in the lungs following aerosol infection occurs rapidly within the first 14 days while bacteria in the spleen remain undetectable. Bacteria become detectable in the spleen at 3 weeks, indicative of dissemination from the lungs to peripheral organs. Furthermore, bacterial load in the lungs becomes maximal at the 3-week time point and stabilizes at subsequent time points. Colony counts are maintained at this level out to 8 weeks post-infection (data not shown). Animals were also skin tested with PPD as an indicator of systemic cell-mediated immune responses. Positive skin test conversion was observed in all animals tested at 3 weeks post-infection concomitant with extra-pulmonary dissemination of bacteria. This time frame of dissemination and PPD conversion response are consistent with previous reports using the guinea pig tuberculosis animal model (33). Thus our animal model system is similar to others that have been described and provides a system in which to test vaccine preparations.



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Fig. 1. Kinetics of M. tuberculosis dissemination and PPD responses. Twelve non-immunized guinea pigs were infected using the nose-only exposure system. Groups of three animals were sacrificed at various time points after infection. The data are expressed as the mean bacterial load in the right caudal lobe of the lung and the whole spleen. The PPD skin test was performed 2 days prior to the time points indicated and read at the time of sacrifice. No detectable c.f.u. was observed in the spleen at 2 weeks after infection, while the lung shows uncontrolled growth for the first 3 weeks after which time the bacterial load is stabilized. At 3 weeks post-infection, bacterial c.f.u. are detected outside the lung as evidenced by the spleen c.f.u. at day 21. The PPD skin tests are uniformly negative at 2 weeks post-infection, but become positive at the time of dissemination of the bacteria from the lung. The day 0 point represents the c.f.u. counts and PPD response from naive animals.

 
Liposome-formulated vaccine preparations
Several lipid antigens presented by CD1 have been identified by analysis of human T cell lines (811). However, these specific antigens identified previously may only represent a portion of the entire population of possible CD1-presented antigens. Therefore, we wanted to immunize animals with a broad array of lipid and glycolipids to stimulate as many lipid-specific T cells as possible. To accomplish this, a lipid extract was generated from M. tuberculosis bacteria by solubilization in chloroform:methanol. Since our goal in the current study was to evaluate the protective response induced by mycobacterial lipid or glycolipid molecules in the absence of concurrent immunization with M. tuberculosis protein antigens, we examined the lipid extract for contamination by protein.

We found that for these studies, the most sensitive and specific means of detecting specific protein contamination was by silver staining of polyacrylamide gels (limit of detection <10 ng for an individual protein of discrete size). Figure 2(A) shows a silver-stained gel of the whole lipid extract used for vaccine formulation. The gel was loaded with 100 µg of lipid extract (Fig. 2A, lane 3). The whole culture filtrate protein fraction and purified Ag85A were used in parallel gel lanes to characterize and estimate the level of any possible contamination (Fig. 2A, lanes 1 and 2). To retain possible small peptides on the gel, if present, we terminated electrophoresis prior to migration of the dye front off the gel. Silver staining of the gel revealed no visible bands in the whole lipid preparations.



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Fig. 2. Biochemical analysis of M. tuberculosis lipid extracts and vaccine formulations. Lipid extracts from M. tuberculosis H37Rv were analyzed by SDS–PAGE and silver staining to assess protein contamination (A): lane 1, 10 µg Ag85A; lane 2, 10 µg culture filtrate proteins; lane 3, 100 µg H37Rv lipid extract. TLC of lipid extract (B): lane 1, 200 µg H37Rv lipid extract; lane 2, 5 µg M. phlei GMM; lane 3, 5 µg mycolic acid; lane 4, 10 µg phosphatidylcholine; lane 5, 10 µg phosphatidylinositol; lane 6, 10 µg phosphatidylethanolamine. Transmission electron microscopy of extruded liposome vesicles made with QS-21 only (C) or QS-21 plus M. tuberculosis lipid extracts (D) showed similar vesicle sizes for both vehicle and lipid vaccine formulations. Bar = 300 nm.

 
We next characterized the lipid molecules present in the chloroform:methanol extracts using TLC followed by staining with cupric acid and charring to detect all organic molecules. Figure 2(B, lane 1) shows the profile of an extract used for immunization of guinea pigs. Numerous lipid species were observed in the whole lipid extract using this technique. Known lipid molecules were run on the same plate for comparison (Fig. 2B, lanes 2–6). These include the CD1-presented antigens GMM and mycolic acid (Fig. 2B, lanes 2 and 3 respectively). A spot in the whole lipid extract that co-migrated with the GMM was detected with this technique. The presence of this antigen in the M. tuberculosis chloroform:methanol extract was confirmed using the CD1b-restricted GMM-specific T cell line LDN5 (10). The lipid extract was capable of stimulating the LDN5 T cell line in a dose-dependent manner (data not shown), thus confirming the presence of GMM in the material used for vaccine preparation.

Based on the hydrophobic nature of the compounds that we isolated, we hypothesized that liposome vesicles would provide an ideal delivery vehicle for lipid antigens. Preliminary studies showed that liposomes were capable of forming in the presence of mycobacterial lipids and that the adjuvant QS-21 could also be incorporated into the vesicles. Vesicles containing M. tuberculosis lipid extracts and adjuvant were generated with a syringe extruder using DSPC and cholesterol as carrier lipids. Electron microscopy was used to estimate the size of the extruded vesicles. Figure 2(C) shows typical liposome vesicles generated from the extrusion of the carrier lipids plus QS-21 adjuvant alone. Similar-sized vesicles were obtained when carrier lipid was extruded in the presence of QS-21 adjuvant plus M. tuberculosis lipid extract (Fig. 2D). Thus, the presence of bacterial lipid components does not appear to affect the integrity or size of liposome vesicles. Moreover, the recovery of total mycobacterial lipids from liposomes was ~77% upon re-extraction of vaccine preparations, based upon the incorporation and re-extraction of radiolabeled mycobacterial lipids (data not shown).

Reduction of bacterial burden in lipid-vaccinated animals
In parallel studies it was found that a lipid extract formulated with QS-21 adjuvant into liposomes was capable of inducing CD1-restricted T cell responses in guinea pigs (28). Preliminary vaccine studies were carried out to determine an effective immunization protocol for M. tuberculosis challenge studies. We tested several parameters including dose and number of immunizations (data not shown). Guinea pigs were injected 3 times with either vehicle liposomes plus adjuvant as a negative control group or vehicle liposomes plus adjuvant with M. tuberculosis lipids incorporated. The positive control group received a single dose of live BCG vaccine. A 7-week rest period was incorporated into the protocol between the last booster immunization and the challenge infection to allow non-specific immune responses to wane. We monitored animals for any adverse reactions to the vaccine preparations following injection. No obvious toxicity or local skin responses were noted. In addition, PPD skin tests were conducted ~1 week prior to challenge infection. No response to intradermal PPD testing was evident on animals that received either vehicle or lipid vaccine preparations, further supporting the absence of immunogenic proteins in the lipid antigen preparations. In contrast, all BCG-vaccinated animals had positive PPD skin test conversions with induration >10 mm in diameter. Interestingly, intradermal skin tests with a 10 µg dose of the lipid extract used for vaccinations in a PBS vehicle failed to generate a dermal delayed-type hypersensitivity reaction in immunized animals. This result suggests that unlike protein antigens, lipid antigens do not induce delayed-type hypersensitivity reactions after intradermal injection.

An initial vaccine experiment was conducted with various lipid extracts without the use of added adjuvants. However, in the absence of adjuvant, no statistically significant reduction of bacterial colony counts were observed with lipid extracts when compared to vehicle control formulations (data not shown). In further studies, we found that formulating M. tuberculosis lipids with QS-21 alone or in combination with other adjuvants was capable of eliciting a reduction in bacterial burden in the lungs. Table 1 shows the bacterial burden from the lung and spleen from three separate vaccine experiments. Bacterial counts from the lipid-vaccinated group show a statistically significant reduction when compared to the control group receiving only vehicle lipids plus adjuvant when the adjuvant QS-21, with or without DDA, was included in the formulation (experiments 2 and 3). There was no statistically significant reduction in bacteria when DDA alone was used as the adjuvant for lipid vaccine immunization (experiment 1). In experiment 3, the BCG-vaccinated group showed a statistically significant reduction of c.f.u. in both the lung and spleen. Because viable BCG bacteria are effectively cleared from guinea pig tissues over the time frame of these experiments (10 weeks), the c.f.u. counts in Table 1 reflect M. tuberculosis bacteria and not residual BCG (33). In all experiments, the number of bacteria in the spleens of lipid-vaccinated animals was also lower when compared to the corresponding vehicle control groups (Table 1). However, the standard deviations tended to be slightly higher in the spleen than in the lung so that statistical significance was not achieved. We also titered the dose of lipid, and tested 10, 1 and 0.1 mg lipid using a constant vehicle and adjuvant mixture. We found that only the 1 mg dose provided statistically significant reduction of bacteria in the lung (data not shown). It is likely that the 10 mg lipid dose did not form vesicles properly, thus reducing the efficacy of the formulation. Taken together, these data indicate that lipid immunization is able to elicit a modest, but statistically significant, reduction of bacteria in the lungs.


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Table 1. Bacterial load in lungs and spleena
 
Reduction of lung pathology in lipid-vaccinated animals
Necropsy of all animals was performed at 4 weeks after aerosol challenge with M. tuberculosis. Gross examination of the lungs revealed significant differences between the vehicle control group and the animals receiving either lipid vaccine or BCG. Figure 3(A) shows representative lung lobes from each of the three experimental groups from vaccine experiment 3. Lungs from animals that received only vehicle plus adjuvant had visibly larger lesions upon inspection, while both the lipid- and BCG-vaccinated animals had smaller lesions that tended not to penetrate the outer surface of the lung tissue. Cross-sections of the lung lobes (Figs 3B and 5A–F) revealed findings consistent with the gross pathology, with the vehicle control group having visibly larger lesions and a higher frequency of lesions with caseous necrotic centers. In contrast, both the lipid vaccine group and the BCG group had smaller lesions that were more compact, with less caseous necrosis.



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Fig. 3. Reduced pathology of M. tuberculosis lesions from the lungs of lipid- and BCG-vaccinated guinea pigs. (A) Gross pathology of representative left caudal lobe from each treatment group from a vaccine trial. Note that the lungs from animals receiving the lipid vaccine or the BCG vaccine had smaller visible primary granulomas than the vehicle control lung. (B) Cross-sections of lung lobes stained with hematoxylin & eosin from each of the three treatment groups shown in (A). Note that the vehicle control group lung section had larger granulomas than the sections from either the lipid vaccine- or BCG-vaccinated animal lung sections. More frequent lesions with caseous necrotic centers were evident in the vehicle control lung section in (B) (arrows).

 
We next used morphometric analysis to quantify the extent of diseased tissue in the lungs of animals from vehicle control and vaccine groups. Whole lung cross-sections (Fig. 3B) from five individual animals from each treatment group were used for analysis. We used the same lung lobe for all histological analysis and oriented tissue similarly for sectioning so that the same anatomical location was sampled for all animals. Figure 4(A) shows that the percentage of diseased tissue in the vehicle control group was approximately twice that of the lipid-vaccinated or BCG groups. This appeared to be due directly to the larger size of the lesions and not to an increased number of lesions in the lung space, as no statistically significant difference in the number of lesions between the three treatment groups was noted (Fig. 4B). Figure 4(C) shows that the mean size of lesions in lipid-vaccinated or BCG groups was less than half that of the vehicle control group. Thus, many of the histological characteristics observed in the lung lesions from lipid-vaccinated animals are also seen in BCG-vaccinated animals, including smaller lesions that are more lymphocytic and less necrotic. Taken together, these histological data suggest that the lipid-vaccinated animals have reduced lung pathology as evidenced from the characteristics of the granulomatous lesions.



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Fig. 4. Morphometric analysis of lung pathology. The size of individual lung lesions was determined from hematoxylin & eosin stained left-caudal lobe cross-sections (Fig. 3B) using computer-assisted image analysis. These data were used to calculate the percent area of lung tissue occupied by diseased tissue. The percentage of diseased tissue was significantly smaller in mycobacterial lipid- and BCG-vaccinated animals when compared to the vehicle control group (A). The differences between the treatment groups are not due to differences in total number of lesions as there is no statistical difference in the number of lesions in each group (B). The size of the lesions is also smaller in mycobacterial lipid- and BCG-vaccinated animals when compared to the size of lung lesions from vehicle control group animals (C). Data are the mean and error bars are SD derived from a sampled lung cross-section of five individuals from each treatment group. A two-tailed Student’s t-test was used to determine statistical significance. Brackets above bars indicate statistical significance (P < 0.05) between the groups with calculated P values shown above the brackets.

 
A more detailed histological analysis of the granulomatous lesions was carried out to further characterize the differences observed between vaccinated and vehicle control groups. Granulomatous lesions were typically smaller and less necrotic in lipid vaccine- and BCG-treated animals (Fig. 5A–F). Closer inspection revealed that the lung lesions from vehicle control animals were less compact in the area surrounding the caseous center on the lesion (Fig. 5G and J). In addition, fewer nuclei are present and this region appears less cellular with numerous vacuolar spaces. In contrast, lung lesions from both BCG- and lipid-immunized guinea pigs show numerous lymphocytes in the lesion (Fig. 5H, I and K). Immunohistological staining revealed approximately equal numbers of CD4+ and CD8+ cells (data not shown). These lesions also contain numerous monocytoid or macrophage-like cells that remained more intact when compared to vehicle control lesions. Lymphocytic infiltrates present in the lungs of BCG-vaccinated animals were often associated with small blood vesicles (perivascular cuffs). Figure 5(L) shows a cuff of lymphocytes surrounding a small vessel with several lymphocytes adherent to the lumenal wall of the vessel. These cuffs were also present in lipid-vaccinated animals, but at a lower frequency. Taken together, the pulmonary histology of lipid-vaccinated animals showed strong similarities with the features found in the BCG-vaccinated animals, suggesting an active process of antigen-induced protection related to the T cell-dependent immunity that is known to result from BCG vaccination.



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Fig. 5. Histology of granulomatous lesions in the lung. Hematoxylin & eosin lung sections from guinea pigs that received either vehicle control (A, D, G and J), lipid vaccine (B, E, H and K) or BCG vaccine (C, F, I and L). (A) Low magnification view of a typical granuloma from a vehicle control animal. Note the caseous necrotic center (black arrow) of the lesion. (B and C) Granulomas from lipid-immunized and BCG-immunized animals respectively. These lesions are typically smaller than the vehicle control lesions. (D) Higher-power view of a necrotic center (black arrow) in a vehicle control animal lung. (E) More compact and defined lesion in a lipid-immunized animal. Lymphocytes are visible in the periphery of the lesion (white arrow). (F) A lesion from a BCG-vaccinated animal showing a large lymphocytic aggregate (white arrow) within the lesion. Perivascular lymphocytic infiltrates were more common in BCG- and lipid-immunized animals. (G and J) Low- and high-power views respectively of the region surrounding the central necrosis. Note the more acellular appearance of vehicle control lesions with fewer lymphocytes compared to the lipid- and BCG-vaccinated animals (H, I, K and L). The black arrow in (G) marks the outer right edge of the necrotic center. (H and K) Low- and high-power views of granulomas from a lipid-immunized animal showing dense projections of lymphocytes (white arrow) into the lesion. Monocytoid or macrophage-like cells remain largely intact within these lesions. (I and L) Low- and high-power views respectively of a lesion from a BCG-immunized animal showing similar histological features to lipid-immunized animals with more lymphocytic infiltrates and a more compact appearance to the lesions. (L) A typical lymphocytic aggregate in a BCG lesion with an associated vessel. Lymphocytes attached to the lumen of the vessel are visible (arrow). Scale bars: 500 µm (A–C); 250 µm (D and E); 125 µm (F); 50 µm (G–I); 25 µm (J and K); 16 µm (L).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The recent appreciation that bacterial lipids and glycolipids can function as T cell antigens requires that these molecules be assessed in the context of a vaccine formulation. Several mycobacterial lipid and glycolipid antigens are recognized by specific T cells in humans. These data point to the possible application of M. tuberculosis lipid molecules as components in a subunit vaccine formulation. Previous studies using the guinea pig tuberculosis model have shown that effective vaccination of animals results in a reduced bacterial burden in the lungs and spleen relative to unvaccinated control animals (32,34). Moreover, the reduced number of bacteria in vaccinated animals measured at early time points was found to correlate with long-term survival (32). Previous studies have also documented reduced pulmonary pathology following effective vaccination (23). Thus, bacterial load and histological evaluation was carried out for each animal in these studies. Initial experiments explored the methodology for administration of mycobacterial lipids including the optimal dose of lipid antigens and the type of adjuvant needed to elicit protection. We found that DDA as an adjuvant did not give reproducible effects and was subsequently dropped from the immunization protocol. It is possible that DDA failed to incorporate into the liposome carrier, but we have no direct evidence of this. Subsequently, we found that liposomes used as a delivery vehicle for the mycobacterial lipids with QS-21 incorporated as adjuvant provided partial protection.

The effects of vaccination on animals were assessed by demonstrating a reduction in bacterial load and quantitative evaluation of pulmonary histopathology. While lipid-immunized animals demonstrated reduced bacterial load, particularly in the lungs, bacterial loads in the spleen were more variable. In general, we observed ~0.3 log decrease in the lung c.f.u. of lipid-immunized animals and no statistically significant decreases in the spleen. In other experiments, BCG typically provided a larger reduction of c.f.u. in the lung (~1 log) and spleen (~2 log), which is more typical of the published values of this vaccine (data not shown). While previous studies have correlated lower bacterial colony counts with improved survival (32), long-term survival was not determined in the present study. However, our results suggest that the immune response induced by lipids may be localized more to the lung than in other tissues. Alternatively, the lipid-mediated effect observed here may be confined to the early phases of the infection and thus have more of a role in the lung, which is the initial site of infection in our aerosol model.

More striking than the decrease in bacterial load were the obvious differences in lung granuloma pathology between vehicle control and lipid-vaccinated animals. Lung granulomas in the vehicle control animals were typically larger, less compact and often had caseous necrotic centers in the lesions. In contrast, the lesions of lipid- and BCG-vaccinated animals were smaller, less necrotic and more lymphocytic. The presence of lymphocytic infiltrates in and around the granuloma suggests that active recruitment of lymphocytes with a capacity to contain bacteria in compact and well-formed lesions occurred before excessive growth of the organism and subsequent immunopathological lung destruction. Since morbidity and mortality in small animal models of tuberculosis results primarily from destruction of lung function by consolidation of inflammatory lesions, the smaller lesions present in lipid-vaccinated animals may indicate an improved prognosis compared to vehicle control animals. Taken together, these data support a role for lipids in the induction of a protective immune response to tuberculosis.

Recent studies in the guinea pig have suggested that, for some vaccines, long-term survival may be improved in the absence of bacillary load differences at early time points (23,35). This may be due to differences in the T cell effector functions elicited by the different immunogens present in a particular vaccine. For example, lipid-induced T cells may produce chemokines that draw lymphocytes into the lesions, but fail to activate bactericidal effector functions in macrophages, thus limiting the size of the lesion without inhibiting bacterial growth. However, there is no direct evidence for such phenomena in the context of tuberculosis infection. This is in contrast to historical data that shows reductions in early bacillary load positively correlate with improved survival in the guinea pig model of tuberculosis (32). These studies were performed comparing BCG and a killed bacterial preparation of M. tuberculosis. In our studies we have examined the bacterial load and pathology at an early time point after challenge infection consistent with the traditional assessment of efficacy in the vaccine model described by Wiegeshaus et al. (32). However, we have not directly assessed long-term survival of animals receiving lipid formulations.

Because of the current lack of methods to eliminate or block guinea pig CD1 function in vivo, we are unable to definitively attribute the protective efficacy of our lipid-based vaccine to CD1-restricted antigen presentation in these experiments. However, we have attempted to minimize other possible explanations for the protective response seen in the mycobacterial lipid-vaccinated animals. For example, some mycobacterial lipids are known to non-specifically activate macrophages and we were concerned that these non-specific effects may account for short-term protective immune responses that do not represent memory immune responses. This concern has been minimized by the incorporation of a 7-week interval between the last immunization and the challenge infection to allow non-specific activation effects to wane (36). In addition, we have recently demonstrated that guinea pigs immunized with mycobacterial lipid extracts identical to those described here were capable of stimulating CD1-restricted T cell responses in vivo (28).

Another concern in these studies was the possible contamination of the M. tuberculosis lipid extracts with peptides that could elicit T cell responses through MHC class I or II pathways. The lipid extraction procedure used in these studies selectively isolates non-covalently associated cell wall lipids. Furthermore, we assessed protein contamination by PAGE and silver-staining methodologies, and by analysis of amino acid content of acid hydrolyzed samples. Protein bands were not detected using PAGE analysis. Amino acids were detected in the extract at levels that did not exceed 1% by weight. However, this amount was unlikely to reflect protein contamination since ~50% of the total amino acids were alanine and glutamate/glutamine, probably originating from mycobacterial peptidoglycan rather than proteins. Therefore we estimated a total protein contamination of <=0.5% by weight per dose. Previous challenge studies using immunization of protein vaccines typically have used doses of >=100 µg (22,23,37). These data indicated that protein contamination in our lipid antigen preparations was unlikely to be relevant to the lower colony counts or improved pathology observed.

In considering the constituents of a subunit vaccine for a particular microbial pathogen, lipid antigens have not been included as possible antigens for stimulating adaptive immune responses. This is because lipid and glycolipid antigens were not previously considered as T cell antigens until the appreciation of the CD1 antigen presentation pathway. For some pathogens, group 1 CD1-restricted T cell responses may provide critical effector functions that augment MHC-restricted T cell responses. This augmentation may be important in determining the outcome for some types of infections. Because of the extensive data already available for several group 1 CD1-presented lipid antigens from M. tuberculosis, we chose to use this bacterial infection model to test the efficacy of lipids antigens as vaccine components. However, it is likely that other organisms possess lipids or glycolipids that can be recognized by specific CD1-restricted T cells and would also contribute to protective immune responses (38). Thus, the data presented here may be more broadly applicable to vaccine development for a wide range of infectious disease pathogens. Moreover, it is likely that the significant responses noted here to a crude lipid extract might result in greater responses to preparations enriched in particularly immunogenic lipid antigens.

Vaccines against infectious pathogens remain one of the most effective and efficient means of disease prevention available. However, non-living vaccine preparations often fail to induce the same level of protective immunity when compared to a live vaccine, especially for intracellular pathogens such as Listeria monocytogenes or M. tuberculosis (39). This is most likely a result of the failure of exogenously delivered protein antigens to reach the cytosol, and be processed and transported to the endoplasmic reticulum in the absence of a natural intracellular infection. The delivery of antigens by liposome vesicles has been shown to enhance induction of cytotoxic T cell responses, presumably by introducing antigens directly into the cell cytosol after fusion with the host cell membrane (40). In addition, lipid antigens themselves may have the capacity to traffic more freely between intracellular membrane compartments (41) and to partition directly into the cellular membranes of an antigen-presenting cell (42). Thus, lipid antigens administered as vaccines may be more effective than proteins in eliciting T cells with appropriate cytolytic effector functions based in part on their biophysical properties. In addition, unlike the MHC molecules, CD1 isoforms are non-polymorphic between individuals. Moreover, the bacterial lipids that are presented by CD1 are generally not subject to major structural changes. These features make lipid antigens that are presented by CD1 attractive targets for vaccine design.

It is well established that presentation of peptides by the MHC class I and II systems is important for an effective cell-mediated immune response against M. tuberculosis infection (5,6). In addition, the CD1-restricted T cells have effector functions consistent with an anti-microbial activity (11,15,43). In considering novel approaches to subunit vaccine development, the T cell responses elicited by protective protein antigens might be augmented by the presence of additional T cell subsets with complementary effector functions. A number of promising protein-based tuberculosis vaccine candidates have already been described (22,23,44). Thus, combining both protein and lipid antigens may provide better protection than either type of antigen alone. Together, these data provide the foundation for further investigation of the practical utility of lipid antigen vaccination for prevention of tuberculosis and other infectious diseases.


    Acknowledgements
 
The authors would like to thank the staff at the Dana Farber Cancer Institute Animal Resource Facility for the excellent care of the animals used in these studies. We also thank Gilla Kaplan and Liana Tsenova for advice on the nose-only aerosol system, and Tan-Yun Cheng for T cell testing the lipid extracts. This work was supported by the Sequella Foundation through a Vaccine Initiatives Program grant and by a Small Business Innovation Research grant from the National Institutes of Health (AI40798). S. A. P. is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, and received additional support from grants from the NIH (AI45889 and AI48933) and the Irene Diamond Foundation. K. H. was supported by a fellowship from the Medical Foundation.


    Abbreviations
 
ADC—albumin–dextrose complex

BCG—bacillus Calmette-Guerin

BL3—Biosafety Level 3

DDA—dimethyl-dioctadecylammonium bromide

DSPC—1,2-distearoyl-sn-glycero-3-phophocholine

GMM—glucose monomycolate

PPD—purified protein derivative

TLC—thin-layer chromatography


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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