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The Journal of Immunology, 2002, 168: 1869-1876.
Copyright © 2002 by The American Association of Immunologists

Cell-Mediated Immunity Induced by Recombinant Mycobacterium bovis Bacille Calmette-Guérin Strains Against an Intracellular Bacterial Pathogen: Importance of Antigen Secretion or Membrane-Targeted Antigen Display as Lipoprotein for Vaccine Efficacy1

Leander Grode*, Mischo Kursar*, Joachim Fensterle{dagger}, Stefan H. E. Kaufmann* and Jürgen Hess2,{ddagger}

* Department of Immunology, Max-Planck-Institute for Infection Biology, Berlin, Germany; {dagger} Institute for Medical Radiation and Cell Research, Wurzburg, Germany; and {ddagger} Department of Molecular Therapy, November AG, Erlangen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Live recombinant vaccines expressing defined pathogen-derived Ags represent powerful candidates for future vaccination strategies. In this study, we report on the differential induction of protective cell-mediated immunity elicited by different recombinant Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains displaying p60 Ag of Listeria monocytogenes in secreted, cytosolic, or membrane-attached form for T cell recognition. Anti-listerial protection evoked by the membrane-linked p60 lipoprotein of rBCG Mp60 and that of the p60 derivative secreted by rBCG Sp60-40 were nearly equal, whereas cytosolic p60 displayed by rBCG Np60 failed to protect mice from listeriosis. In vivo depletion of CD4 or CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in anti-listerial protection. In rBCG Sp60-40-vaccinated animals, CD4 T cells predominantly contributed to anti-listerial control as shown by the failure of anti-CD8 mAb treatment to impair the outcome of listeriosis in rBCG Sp60-40-vaccinated mice after L. monocytogenes challenge. Hence, differential Ag display by rBCG influences cell-mediated immunity, which in turn may impact vaccine efficacy due to the different requirements of CD4 or CD8 T cells for pathogen elimination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycobacterium bovis bacille Calmette-Guérin (BCG),3 an attenuated M. bovis strain, offers great potential as a live recombinant carrier delivering foreign protein Ags to be recognized by the immune system. Indeed, BCG is currently the most common human vaccine that has already been used to vaccinate more than three billion people against tuberculosis (TB) (1). Numerous viral, bacterial, and parasitic Ags have been successfully expressed in BCG and were shown to stimulate humoral and cell-mediated immune responses against respective pathogens (2, 3, 4). Moreover, several reports have emphasized the importance of differential Ag display by BCG in the context of Ab-mediated protective immunity (5, 6, 7). Display of relevant Ags (OspA, PspA, and MSP1) as secreted proteins or membrane-associated lipoproteins by rBCG is central for protection against Streptococcus pneumoniae, Borrelia burgdorferi, or Plasmodium yoelii, respectively (5, 6, 7). In infectious disease models with emphasis on CD4 and CD8 T cell-controlled immunity, it remains to be determined whether Ag display by rBCG influences vaccine efficacy due to the lack of protection studies (4, 8, 9, 10).

In contrast, for attenuated recombinant Salmonella typhimurium vaccines, several reports underline the importance of Ag secretion to elicit protective T cell-mediated immune responses against lymphocytic choriomeningitis virus or Listeria monocytogenes infection in mice (11, 12, 13, 14). Both Ag carriers, S. typhimurium and BCG, reside within phagocytic vacuoles inside host cells, thereby sharing several similarities (15). In general, Ags delivered into the phagolysosome are preferentially processed via the MHC class II pathway and, as a consequence, stimulate CD4 T cells predominantly (16). In contrast, Ags with cytosolic localization are processed along the MHC class I pathway to induce CD8 T cells (17). Therefore, intraphagosomal bacteria such as attenuated S. typhimurium or BCG preferentially activate CD4 T cells, whereas bacteria capable of leaving the vacuolar compartment to enter the host cell cytosol additionally induce CD8 T cells (4, 10, 16). One member of this latter group of microorganisms is represented by the Gram-positive rod-like pathogen L. monocytogenes, which causes listeriosis in mice and man (16). Due to the intracytosolic localization of L. monocytogenes within host cells, the listerial protein p60, which is secreted in large amounts into the bacterial environment, represents a prominent source for MHC class I and II epitopes (18, 19, 20). Due to its bifunctional properties during bacterial septation of and host cell entry by L. monocytogenes, the biological role of p60 remains controversial (21, 22). CD8 T cells of H-2Kd haplotype and specific for the epitopes p60217–225 and p60449–457 are capable of adoptively transferring protection to naive BALB/c mice against subsequent challenge-infection with L. monocytogenes (23, 24). Although p60449–457 represents the predominant epitope among listerial MHC class I-associated peptides with up to 10,000 epitopes per cell after 6 h of in vitro infection (25), it elicited a subdominant T cell response in vivo compared with that induced by the p60217–225 epitope (25). Recombinant S. typhimurium secreting p60 fusion proteins via the type-1-secretion machinery induced cell-mediated immunity against murine listeriosis, whereas recombinant S. typhimurium blocked in their capability to secrete this p60 hybrid Ag failed to evoke anti-listerial protection, underlining the highly immunogenic potential of secreted Ags displayed by viable delivery devices (11). This indicates that p60 is a valuable tool for the evaluation of Ag display by rBCG in mice. Therefore, experiments with rBCG strains expressing this immunodominant p60 Ag in different bacterial compartments were performed to quantitatively analyze the contribution of Ag display for protective immune responses. In this study, we report on the superior vaccine efficacy of p60 targeted as lipoprotein derivative to the mycobacterial membrane surface against listeriosis. Furthermore, we show that anti-listerial protection depended on interactions between CD8 and CD4 T cells with Th1-cytokine profile. Our study identifies membrane-anchored Ag display by lipoprotein signal sequences as an important parameter for evoking T cell-mediated protection by rBCG.


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

BALB/c mice were kept under specific pathogen-free conditions in our facilities at the "Bundesgesundheitsamt fur Veterinarmedizin und Verbraucherschutz" (Berlin, Germany) and were fed autoclaved food and water ad libitum. In a given experiment, mice were age and sex matched.

Bacteria and cells

BCG strain Danish 1331 (Statens Serum Institut, Copenhagen, Denmark) was cultured in Dubos broth base (Difco, Detroit, MI) supplemented with Dubos medium albumin (Difco) at 37°C. A mid-logarithmic culture was aliquoted and stored at -70°C until use. L. monocytogenes EGD Sv 1/2a and M3 strains were grown in brain heart infusion broth (Difco) at 37°C with aeration (26). Plasmid pSK-5 was a generous gift of Drs. I. Gentschev and W. Goebel (University of Wurzburg, Germany; Ref. 27). The mycobacteria-Escherichia coli shuttle vectors pAT261, p2619s, and pRB26 were kindly provided by MedImmune (Gaithersburg, MD; Ref. 28). P815 mastocytoma cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 (Life Technologies, Karlsruhe, Germany) supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 U/ml), and 2-ME. This medium is referred to as RP10 medium.

DNA manipulation and sequencing

Extrachromosomal pAT261, pRB26, and p2619s expression plasmids were used for compartmentalized p60 expression by different rBCG strains as described previously (5, 6, 28). The 1-kb PstI-SalI fragment (original PstI-position 942; Ref. 27) of a pUC18-p60 construct harboring a PCR-derived iap gene of L. monocytogenes EGD (used oligonucleotides for PCR: forward-p60-TACCCGGGATCCAGCAAGCACTGTAGTAGTCGA and reverse-p60-ATCCCGGTCGACTTATACGCGACCGAAGCCAA) was applied for constructing pRB26:p60. The complete BamHI-SalI DNA fragment of pRB26:p60 coding for the C-terminal 319 aa of p60 was introduced into the following plasmids: p2619s and pAT261. The correct DNA sequence of the resulting constructs p2619s:p60, pRB26:p60, or pAT261:p60 at the sites of fragment insertion was determined using the following oligonucleotides 2619-GGGTGAAGCGTGGACTGA, RB26-CGATCGCTAGATCCATGG, and AT261-CCGCGACATCAAGGTTCA, respectively (Sequiserve, Vaterstetten, Germany).

Characterization of rBCG strains

The plasmids pRB26:p60, pAT261:p60, or p2619s:p60 were introduced into BCG strain Danish 1331 by a standard electroporation protocol (5, 6, 28) and then r-colonies of rBCG strains Np60, Sp60-40, or Mp60 were selected on Middlebrook 7H10 agar supplemented with kanamycin (15 µg/ml). Kanamycin-resistant colonies were grown to mid-logarithmic phase in Dubos liquid medium (Difco) containing 10% Dubos medium albumin (Difco) and 15 µg/ml kanamycin for 3 wk. The bacteria of each rBCG strain normalized to a CFU of 5 x 109 microorganisms were harvested by centrifugation at 10,000 x g. The pellet was resuspended in PBS and was transferred to a tube filled with glass beads (BIO101; Quantum Appligene, Heidelberg, Germany). The bacteria were ribulized in the FASTprep FP120 machine (BIO101 Savant; Quantum Appligene) at speed 6.5 for 45 s to solubilize them, and they were then centrifuged at 10,000 x g for 10 min. The supernatant referred to now on as the somatic fraction was then stored at -20°C. The pellet was resuspended with PBS/0.05% (v/v) Tween 20, and was again ribulized at the speed of 6.5 for 45 s and was centrifuged at 10,000 x g for 10 min. The supernatant referred to now on as the membrane fraction was then stored at -20°C. The protein content of each preparation was adjusted to 2 mg/ml total protein. According to the manufacturer’s instructions, 20 mg was separated on 10% SDS-PAGE (Bio-Rad, Richmond, CA). The separated proteins were electrophoretically transferred to a Hybond ECL nitrocellulose membrane (Amersham, Little Chalfont, U.K.) which was blocked overnight with PBS containing 1% BSA. The membrane was washed in PBS-Tween 0.05%, and was subsequently incubated with appropriate rabbit Abs specific for p60 diluted in PBS for 2 h. After washing, the membrane was stained with HRP-coupled anti-rabbit IgG (diluted 1/20,000; BD PharMingen, San Diego, CA) for 1 h. The signal development on x-ray film (XOMAT-AR; Eastman Kodak, Rochester, NY) was performed with ECL kit (Amersham) for 30 s up to 1 min (29).

Vaccination of mice with rBCG strains

One group of BALB/c mice was vaccinated i.v. with 106 microorganisms of rBCG strains or BCG control and was sacrificed for CFU analysis 15, 30, and 60 days postvaccination. At indicated time points postvaccination, lung and spleen of these mice were removed and homogenized with a lab blender (Seward Medical, London, U.K.). rBCG microorganisms were enumerated by plating serial 10-fold dilutions in PBS on Middlebrook agar plates (Difco) supplemented with 10% oleic acid albumin dextrose catalase enrichment (Difco) and 15 µg/ml kanamycin. After 3–4 wk of incubation at 37°C, mycobacterial colonies were counted. The other group of rBCG-vaccinated mice were challenge-infected i.v. with 105 L. monocytogenes EGD bacteria (10 x LD50) on day 120 postvaccination, and survival was monitored for up to 10 days postchallenge. On day 10 postchallenge, listerial CFU in spleens of rBCG Mp60- or Sp60-40-vaccinated BALB/c survivors were determined by plating serially diluted organ homogenates on PALCAM-L. monocytogenes-selective agar (Merck, Darmstadt, Germany).

Generation of tetrameric PE-conjugated H2-Kd-p60217–225 peptide complexes

Modified forms of the full-length cDNA of the H2-Kd H chain and human {beta}2-microglobulin were kindly provided by E. G. Pamer. Tetrameric H2-Kd/peptide complexes (p60217–225) were generated following the protocol described previously (30, 31).

Staining and flow cytometry analysis

For flow cytometry analysis, 1 x 106 cells per reaction were added to a 1.5-ml Eppendorf tube. The cells were incubated for 15 min at 4°C with 10 µg/ml Fc block (BD PharMingen), 10 µg/ml ChromPure Rat IgG (Dianova, Hamburg, Germany), and 50 µg/ml unconjugated streptavidin (Molecular Probes, Eugene, OR) in 120 µl of PBS (pH 7.45) containing 0.5% BSA and 0.01% sodium azide. After incubation, the cells were triple stained for 60 min at 4°C with 3 µg/ml Cy-5-conjugated anti-CD8{alpha} mAb (clone 169 rat IgG2b), 1 µg/ml FITC-conjugated anti-CD62L mAb (clone Mel. 14), and 5 µg/ml PE-conjugated H2Kd-p60217–225 tetramers. The cells were then washed three times with PBS (pH 7.45) containing 0.5% BSA and 0.01% sodium azide. Three-color flow cytometry was performed using a FACSCaliber flow cytometer, and data were further analyzed with CellQuest software (BD Biosciences, Mountain View, CA).

In vivo depletion of CD4 and CD8 T cells

BALB/c mice were vaccinated i.v. with 106 microbes of the rBCG Mp60 and Sp60-40 or the parental BCG strain for 120 days, and were then treated with a combination of 200 mg/ml isoniazid (Sigma-Aldrich, St. Louis, MO) and 100 mg/ml rifampicin (Sigma-Aldrich) dissolved in drinking water for an additional 2 wk to clear any remaining mycobacteria. Five days afterward, in vivo depletion of CD4 or CD8 T cell subsets was performed 3 days before and 2 days after challenge infection by i.p. injection of 0.3 mg of either anti-CD4 (clone 191) or anti-CD8{alpha} (clone 169) mAb in a volume of 300 µl of PBS. Control BALB/c animals received 0.3 mg of isotype-specific IgG Ab. To confirm the efficiency of in vivo cell depletion (>=98%), a single cell suspension (105 splenocytes) from anti-CD4- or anti-CD8 mAb-treated mice was prepared for immunostaining with FITC-conjugated anti-CD4 mAb (clone GK 1.5) or Cy-5-conjugated anti-CD8{beta} mAb (clone H35-17.2), respectively. Three-color flow cytometry was performed using a FACSCaliber flow cytometer, and data were further analyzed with CellQuest software (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and localization of p60 from L. monocytogenes in rBCG

To find out the most immunogenic mode of Ag display by rBCG for the induction of protective cell-mediated immunity against murine listeriosis, several rBCG strains were constructed with differential p60 localization. The E. coli-mycobacteria shuttle vectors pRB26, pAT261, and p2619s were used to generate rBCG strains expressing p60 of L. monocytogenes in different mycobacterial compartments (5, 6, 32). A PCR-derived 1-kb PstI-SalI DNA fragment coding for the C-terminal 319 amino acids of p60 (position 166 - 484; Ref. 27) was inserted into the plasmid pRB26, resulting in pRB26:p60. The rBCG construct carrying pRB26:p60 was termed Np60 (Table IGo). Subsequently, the BamHI-SalI DNA fragment of pRB26:p60 was introduced into the vectors pAT261 and p2619s, which were termed now pAT261:p60 and p2619s:p60, respectively (Table IGo). The corresponding rBCG strains harboring these plasmids were called Sp60-40 and Mp60. As outlined in Fig. 1Go, the plasmids pAT261:p60 and p2619s:p60 directed p60 expression via respective signal peptides to the supernatant or to the membrane surface of the rBCG strains, Sp60-40 and Mp60. To characterize p60 expression by the different rBCG strains Np60, Mp60, or Sp60-40, appropriate supernatants, mycobacterial lysates, and membrane preparations of mid-logarithmic-grown cultures were prepared as described in Materials and Methods. In membrane fractions, the p60 hybrid Ag was only detected in Mp60-specific protein preparations at 43 kDa (Fig. 2Go). Moreover, as expected, only the cytoplasmic Ag samples of rBCG strains Np60 and Sp60-40 could be stained with anti-p60 Ab (Fig. 2Go).


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  		Table I. Description of rBCG strains

 


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  		FIGURE 1. BCG/E. coli shuttle expression vectors used to express r-p60 of L. monocytogenes. Plasmid vector pRB26, the parental vector of all vectors used in this study, is composed of expression cassettes encoding for kanamycin resistance (kan), an E. coli origin of replicon (Erep), a mycobacterial plasmid replicon derived from plasmid pAL5000 (Mrep), a multiple cloning site, the hsp60 promoter, ribosomal binding site (RBS), and the initial six codons of the BCG hsp60 gene, as described previously (6 ). Plasmid p2619s represents a derivative of pRB26 and includes a DNA fragment encoding the RBS and 5'-DNA coding sequence for the signal peptide of the 19-kDa lipoprotein downstream from the hsp60 promoter. Plasmid pAT261 carries a DNA segment coding for the 5'-DNA coding sequence for the export signal of Ag85B from BCG. An iap gene segment encoding the C-terminal 34-kDa region of the p60 Ag from L. monocytogenes EGD strain was PCR amplified and cloned into pRB26, p2619s, or pAT261.

 


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  		FIGURE 2. Localization of p60 fusion proteins in rBCG strains. The rBCG lysates and membrane samples were prepared by centrifugation and phase partitioning as described in Materials and Methods. Samples of each rBCG strain were normalized to a CFU of 5 x 109 microorganisms and were subjected to SDS-PAGE, electroblotted to nitrocellulose, and immunostained with rabbit anti-p60 Ab. The control of each fraction is flanked on each site for cytosol of BCG wild-type (left) and membrane BCG wild-type (right).

 
Impact of differential p60 Ag display by rBCG on the specific immune response

The frequencies of epitope-specific CD8 T cells recognizing p60217–225 peptide induced by a single i.v. immunization with rBCG strains were determined on days 12, 30, and 60 postvaccination. To verify the importance of membrane-anchored p60 localization for the strength of the CD8 T cell response, direct ex vivo analysis of p60217–225-specific CD8 T cell frequencies were performed by means of FACS staining with labeled H-2Kd- p60217–225 tetrameric complexes. In four independent experimental kinetics, the highest CD8 T cell frequencies for the p60217–225 epitope were always measured on day 30 postvaccination (Fig. 3Go). At this time point, the proportion of p60217–225 tetramer-positive CD8 T lymphocytes ranged from 1.1 to 1.5, which is similar to data obtained in L. monocytogenes infection experiments at day 7 postinfection (Fig. 3Go) (25).



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  		FIGURE 3. Direct ex vivo TCR staining of p60217–225-specific T cells. A, Kinetics of p60217–225-specific CD8 T cell frequencies from three spleens of BALB/c mice per indicated time point were stained with Cy-5-conjugated anti-CD8{alpha} mAb, FITC-conjugated anti-CD62L mAb, and PE-conjugated p60217–225 tetramers as described in Materials and Methods. The animals were vaccinated i.v. with 106 CFU of rBCG strains or the parental BCG vector. B, Measurement of frequencies of CD8 T cells recognizing p60217–225 by tetramer staining in three independent experiments at day 30 postvaccination. BALB/c mice were immunized i.v. with 106 CFU of Mp60 ({blacksquare}), BCG ({blacktriangleup}), or 103 CFU of L. monocytogenes ({diamondsuit}). Splenocytes of each experimental group were collected from three nonvaccinated or vaccinated mice.

 
Correlation between the immune responses elicited by different rBCG strains expressing p60 and protection against murine listeriosis

To correlate immune effector mechanisms elicited by these rBCG strains with the respective vaccine efficacy, BALB/c mice were immunized i.v. with 106 microorganisms of Np60, Mp60, Sp60-40, or the parental BCG vector and were then challenge-infected with L. monocytogenes. All control mice succumbed to this high listerial challenge inoculum. Vaccination with Mp60 displaying p60 as a membrane-anchored lipoprotein derivative rendered mice fully protected, and Sp60-40 induced 80% protection (Fig. 4Go). The two survival curves of Mp60- or Sp60-40-immunized mice were not significantly different as assessed by log rank statistics. The rBCG strain Np60 and the BCG vector strain failed to protect mice from lethal listeriosis in a statistically significant way (Fig. 4Go). The CFU in spleens of survivors vaccinated with rBCG Mp60 or Sp60-40 were determined at day 10 postchallenge. Interestingly, at this day, Mp60-immunized animals showed a significantly lower listerial load in spleens than mice vaccinated with rBCG Sp60-40 (Fig. 4Go; p < 0.05, unpaired t test).



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  		FIGURE 4. Vaccine efficacy of different rBCG strains against murine listeriosis. A, Survival of five BALB/c mice per group vaccinated i.v. with the rBCG strains Np60 ({triangleup}), Mp60 ({blacktriangledown}), Sp60-40 ({diamondsuit}), or the parental BCG ({square}) after lethal L. monocytogenes challenge (10 x LD50). The survival differences between Mp60- versus Sp60-40-vaccinated groups are not statistically significant (log rank test). B, L. monocytogenes-specific CFU in spleens of Mp60-({blacktriangledown}) and Sp60-40-vaccinated ({diamondsuit}) survivors at day 10 after listerial challenge. The median of CFU results from four or five animals per group is presented. CFU differences between rBCG Mp60- and Sp60-40-treated mice are statistically significant (p < 0.05, unpaired t test). The same experiment was repeated twice with similar results.

 
To determine the contribution of CD4 and CD8 T cells to anti-listerial protection induced by Mp60 or Sp60-40 vaccination, BALB/c mice were depleted from respective T cell subpopulations by anti-CD4 or anti-CD8 mAb treatment 3 days before and 2 days postchallenge infection with 5 x LD50 of L. monocytogenes. These treatments caused >=98% depletion of the respective T cell subset before listerial challenge as assessed by cytofluorometry. To underline the effect of the T cell depletion on anti-listerial immunity, a control group of BALB/c mice was included that was immunized with a sublethal dose of L. monocytogenes and was challenged similarly to the rBCG-vaccinated animals. Anti-CD4 mAb treatment led to the death of nearly all Mp60-, Sp60-40-, or BCG control-vaccinated mice by day 10 postchallenge (Fig. 5Go and Table IIGo). In contrast, animals previously immunized with L. monocytogenes survived the lethal listerial infection after CD4 T cell depletion, suggesting that CD8 T cells were sufficient for protection. In contrast, protective anti-listerial immunity evoked by rBCG Mp60 or by L. monocytogenes vaccination of BALB/c mice was not maintained after depletion of CD8 T cells. The immune response elicited by rBCG Sp60-40 fully protected mice against lethal listeriosis, even after the depletion of CD8 T cells (Fig. 5Go and Table IIGo). In conclusion, p60 secretion by rBCG Sp60-40 predominantly induced protective p60-specific CD4 T cells for listerial control, whereas membrane-anchored p60 lipoprotein displayed by rBCG Mp60 stimulated both p60-specific CD8 T and CD4 T lymphocytes to keep L. monocytogenes infection in check.



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  		FIGURE 5. In vivo depletion of distinct T cell subsets in rBCG-vaccinated animals before and after lethal L. monocytogenes challenge. A, Isotype-specific mAb-treated BALB/c animals immunized with BCG ({blacksquare}), Mp60 ({blacktriangleup}), Sp60-40 ({blacktriangledown}), or L. monocytogenes ({diamondsuit}). B, Anti-CD4 mAb-treated mice vaccinated with rBCG ({blacksquare}), Mp60 ({blacktriangleup}), Sp60-40 ({blacktriangledown}), or L. monocytogenes ({diamondsuit}). C, Anti-CD8 mAb-treated mice vaccinated with rBCG ({blacksquare}), Mp60 ({blacktriangleup}), Sp60-40 ({blacktriangledown}), or L. monocytogenes ({diamondsuit}). BALB/c mice were vaccinated i.v. with 106 microbes of the rBCG Mp60 and Sp60-40 or the parental BCG strain for 120 days and were then cleared from remaining mycobacteria by chemotherapy. Five days later, in vivo depletion of distinct CD4 or CD8 T cell subsets was performed 3 days before and 2 days after challenge infection with 5 x LD50 of L. monocytogenes as described in Materials and Methods. The same experiment was repeated twice with similar results. The differences of survival data between each group (Sp60-40, Mp60, BCG, or L. monocytogenes) and the BCG control group were statistically significant (p < 0.05; log rank test and Kaplan Meier statistics).

 

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  		Table II. Survival after L. monocytogenes infection

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report describes rBCG strains expressing the immunodominant Ag p60 of L. monocytogenes in different bacterial compartments, and correlates induction of cell-mediated immune responses with protection. We found that derivatives of p60 directed to distinct compartments (cytosolic, membrane, and supernatant) were detectable in respective fractions of different rBCG strains; that the persistence of the different rBCG strains was not significantly influenced by p60 expression in vivo; that the p60217–225 epitope-specific CD8 T cell response of rBCG Mp60 peaked on day 30 postvaccination as assessed by tetramer staining; that at day 10 postchallenge, anti-listerial protection induced by rBCG Mp60 was superior to that of Sp60-40 as measured by CFU analysis on d 10; and that in vivo depletion of CD4 and CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in vaccine-mediated protection. In the case of rBCG Sp60-40, CD4 T cells predominantly contributed to anti-listerial control.

The C-terminal part of listerial p60 used for the construction of the rBCG strains carries three immunodominant T cell epitopes of H-2d haplotype (19, 20, 24, 27). Besides the two H-2Kd-specific CD8 T cell epitopes p60217–225 and p60449–457, Ag p60 also carries an immunodominant p60301–312 epitope for H-2I-Ad-restricted CD4 T cells (19, 20, 24). Altogether, p60 of L. monocytogenes represents a versatile model Ag for analyzing CD4/CD8 T cell responses on the clonal level. Experimental infection with L. monocytogenes represents a well-established infectious disease model in which both CD4 and CD8 T cells contribute to cell-mediated protection. Hence, it offers an ideal system for the evaluation of rBCG strains in terms of cell-mediated immunity (16). It is known that live BCG microorganisms induce nonspecific anti-bacterial activity in mice, which is mediated mainly by inflammatory cytokines and nitric oxide that is protective against a lethal L. monocytogenes challenge within the first week after BCG vaccination (33). For this reason, we selected a vaccination schedule for the evaluation of anti-listerial protection by rBCG:p60 with vaccination-challenge intervals of 120 days.

To our knowledge, this is the first report in which the mycobacteria-E. coli shuttle vectors pRB26, p2619s, and pAT261 with extrachromosomal replication modes were compared for the evaluation of cell-mediated immunity induced by different rBCG strains due to the genetic consistency of used plasmid constructs. Although we are aware that p60 expression in distinct compartments of these rBCG strains was not analyzed quantitatively, the consistent usage of the pAL5000 replicon and the hsp60 promoter by the corresponding vectors allow the immunological evaluation of different rBCG strains in terms of Ag compartmentalization (3). Moreover, we believe that in vitro quantification of rAg expressed by rBCG would not reflect the actual quantity of rAg available in vivo (34). Our report describes the different qualities of cell-mediated immunity demonstrated especially by the rBCG strains Sp60-40 and Mp60, which may not only be due to different amounts of Ag available for immune recognition because Ag secretion by rBCG may result in increased Ag abundance rather than a membrane-anchored acylated Ag expression (34).

As already outlined by Köhler et al. (27), the high positive charges of the p60 fusion proteins of rBCG Np60, Mp60, or Sp60-40 can explain the considerable discrepancies between the experimentally determined sizes and the calculated molecular masses. It should be noted that p60 of L. monocytogenes itself, with a calculated molecular mass of 50.7 kDa, has an experimentally determined size of 60 kDa (27).

As reported by different groups, the secretion of rAg by rBCG was not superior to cytoplasmic rAg expression in the generation of CD4 and CD8 T cell responses in mice, guinea pigs, and rhesus monkeys (28, 35, 36, 37, 38). Yet, our comparative study revealed a strong impact of Ag compartmentalization by rBCG on anti-listerial protection because only vaccination with rBCG Sp60-40 secreting the 43-kDa p60 fusion protein protected against murine listeriosis predominantly mediated by CD4 T cells. The prominent participation of CD4 T cells in protection of rBCG Sp60-40-vaccinated mice was confirmed by depletion of immune CD8 T cells before L. monocytogenes challenge. These animals survived lethal listeriosis, suggesting a dominant contribution of CD4 T cells to protective immunity without CD8 T cell requirement. Consistent with this finding, p60217–225 tetramer-positive CD8 T cells were not significantly induced in rBCG Sp60-40-vaccinated mice up to 60 days postinfection. This finding is in line with anti-listerial protection of naive BALB/c mice adoptively conferred by IFN-{gamma}-secreting Th1-biased CD4 T lymphocytes specific for the p60301–312 peptide (20). Recently, several subdominant p60 epitopes were identified by novel ex vivo ELISPOT analysis of separated T cell populations from L. monocytogenes-infected BALB/c mice, which could additionally contribute to anti-listerial protection induced by rBCG Sp60-40 (39).

Our report describes the impact of membrane-anchored acylated Ag display on anti-listerial immunity induced by rBCG Mp60. Mice vaccinated with rBCG Mp60 and depleted of immune CD4 or CD8 T cells before challenge were not protected against L. monocytogenes infection. In contrast to rBCG Sp60-40-mediated protection, only a combination of CD4 and CD8 T cells improved the outcome of listeriosis after rBCG Mp60 vaccination. As evidenced by CD4 T cell depletion, it could be suggested that CD4 T cell priming was more potent in rBCG Sp60-40-vaccinated than in Mp60-immunized animals. The apparently lower stimulation of p60-specific and -protective CD4 T cells by rBCG Mp60 immunization of mice could be possibly described by the following two explanations: 1) loss of p60-derived epitopes from MHC class II-loading compartments lead to the impaired stimulation of p60-specific CD4 T cells, as previously described for listeriolysin of L. monocytogenes (40), due to subcellular trafficking of the acylated p60 fusion protein within APC; or 2) involvement of acylated 19 kDa in inhibition of Toll-like receptor 2-dependent down-regulation of MHC class II Ag processing by macrophages (41). We favor the first notion for the failure of CD4 T cell induction by rBCG Mp60 vaccination because only the coding region for the 19-kDa signal peptide and not the whole open reading frame of the 19-kDa protein was used for the generation of the rBCG Mp60 strain. The question remains open whether the lipid tail of the 19-kDa protein alone could inhibit MHC class II processing of macrophages (41). In three independent experiments, we observed similar frequencies of p60217–225 tetramer-positive CD8 T cells in mice after infection with L. monocytogenes or rBCG Mp60 at days 15, 30, or 60.

In general, the mechanisms by which lipoprotein derivatives of rBCG evoke such a broad spectrum of immune responses are still to be elucidated. Note that immunization of mice with peptides conjugated to a palmitoyl chain stimulated increased CD4 and CD8 T cell responses without adjuvants in a wide range of MHC class I and II haplotypes (42). It is also well known that the expression of foreign Ag with the signal sequence of the 19-kDa lipoprotein of M. tuberculosis improves the induction of humoral immunity in mice. This is consistent with a B cell-activating capacity of these lipid residues (5, 6). Recently, the potential immunostimulatory effects of the 19-kDa lipoprotein were also shown for the generation of T cell responses as indicated by markedly increased IL-12 production via Toll-like receptor engagement in human macrophages (43). The acylated 19-kDa Ag of M. tuberculosis expressed by the nonpathogenic host strain rMycobacterium vaccae resulted in the induction of strong Th1-biased immune responses to the 19-kDa Ag (44). However, challenge experiments of immunized mice with M. tuberculosis did not provide evidence for protection by the 19-kDa protein, and the presence of the rAg 19 kDa abrogated the limited protection conferred by the M. vaccae vector strain (44). Yet, other experiments identified the 19- and 38-kDa lipoproteins of M. tuberculosis as prominent sources for MHC class I-restricted epitopes in mice and humans and, hence, support the notion of immunological relevance of these Ags (45, 46, 47). Furthermore, a recent study revealed that peptides derived from the 19-kDa protein or 19-kDa fusion protein have direct access to the MHC class I presentation pathway via TAP-independent mechanisms (48). Electron microscopy and subcellular fractionation of BCG-infected bone marrow-derived macrophages suggested that the acylated and membrane-linked 19 kDa-protein is exported from the mycobacteria-containing phagosome during the first hour after phagocytosis of microorganisms (48). Subcellular trafficking of 19-kDa protein derivatives expressed by different rM. vaccae strains depended on acylation and was independent from glycosylation of the secreted 19-kDa lipoprotein (48). A causal link between this lipid component attached to the amino-terminal cysteine residue of fusion proteins carrying the 19-kDa-derived signal peptide and elevated MHC class I presentation of a 19-kDa Ag fusion with the influenza virus nucleoprotein (NP) was demonstrated by the failure of a nonacylated 19-kDa-NP mutant to stimulate NP-specific CTL after infection of bone marrow-derived macrophages with rM. vaccae expressing the appropriate 19-kDa-NP hybrid construct (48). In summary, only acylation of a mycobacterial surface-attached and intracellularly secreted 19-kDa Ag fusions promoted induction of Ag MHC class I-restricted CTL via intracellular trafficking of these lipoproteins in bone marrow-derived macrophages and dendritic cells (48).

In general, due to the limited knowledge about immunodominant mycobacterial Ags and the lack of highly efficacious adjuvants for humans, which could in combination induce protective cell-mediated immunity against TB, improvement of BCG remains the best choice for rational design of TB vaccines (49). Our study provides new insights into the importance of targeting Ags unique for the proteome of M. tuberculosis to highly immunostimulatory compartments of BCG. We learned from our study that Ag compartmentalization by rBCG apparently influenced distinct cellular branches of cell-mediated immunity. Many groups have now demonstrated the importance of anti-mycobacterial CD8 T cells besides the well-known participation of CD4 T cells in TB control (50, 51, 52, 53, 54). For achieving protective cell-mediated immunity with emphasis on CD4 and CD8 T lymphocytes against TB, we consider Ag display as membrane-anchored lipoprotein, as well as secreted protein, to be the combined and most immunodominant mode for mycobacterial Ag delivery by improved BCG vaccines.


   Acknowledgments
 
We thank M. Hess, D. Miko, J. Hellwig, and L. Lom-Terborg for fruitful discussions and expert help. We are grateful to MedImmune and Dr. W. R. Jacobs, Jr., to Drs. I. Gentschev and W. Goebel, to Dr. D. B. Young, and to Dr. E. G. Pamer for kindly providing mycobacteria-E. coli shuttle plasmids, plasmid pSK5, rabbit anti-19-kDa Abs, and H-2Kd H chain and human {beta}2-microglobulin, respectively.


   Footnotes
 
1 This work was supported by the German Research Society Deutsche Forschungsgemeinschaft Ka 573/3-2-1-2 and European Community cluster "TB Vaccine" (QLRT-PL1999-01093 to J.H. and S.H.E.K.). Back

2 Address correspondence and reprint requests to Dr. Stefan H. E. Kaufmann, Department of Immunology, Max-Planck-Institute for Infection Biology, D-10117 Berlin, Germany. E-mail address: kaufmann{at}mpiib-berlin.mpg.de Back

3 Abbreviations used in this paper: BCG, Mycobacterium bovis bacille Calmette-Guérin; TB, tuberculosis; NP, nucleoprotein. Back

Received for publication June 26, 2001. Accepted for publication December 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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