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Infection and Immunity, July 2004, p. 4109-4113, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4109-4113.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Protection Afforded by Heat Shock Protein 60 from Francisella tularensis Is Due to Copurified Lipopolysaccharide

M. G. Hartley,^1* M. Green,¹ G. Choules,¹ D. Rogers,¹ D. G. C. Rees,¹ S. Newstead,¹ A. Sjostedt,² and R. W. Titball^1,3

Defence Science and Technology Laboratory, Porton Down, Salisbury, Wilts SP4 OJQ,¹ Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom,³ Department of Clinical Bacteriology, Umea University, S-901 85 Umea, Sweden²

Received 10 October 2003/ Returned for modification 12 January 2004/ Accepted 31 March 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heat shock proteins (Hsps) have attracted significant attention as protective antigens against a range of diseases caused by bacterial pathogens. However, more recently there have been suggestions that the protective response is due to the presence of peptide components other than Hsps. We have shown that mice that had been immunized with purified heat shock protein 60 (Hsp60) isolated from Francisella tularensis were protected against a subsequent challenge with some strains of the bacterium. However, this protection appeared to be due to trace amounts of lipopolysaccharide, which were too low to be detected by using the Limulus amoebocyte lysate assay. This finding raises the possibility that the protection afforded by other bacterial Hsp60 proteins may be due to trace quantities of polysaccharide antigens carried by and acting in conjunction with the Hsps.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heat shock proteins (Hsps) are molecular chaperones involved in the folding, unfolding, and translocation of polypeptides. They are highly conserved, ubiquitous proteins, expressed in both prokaryotic and eukaryotic cells. They are constitutively expressed at low levels in unstressed cells, but are up-regulated in response to environmental stress such as heat. In pathogenic bacteria, they are thought to be maximally induced during the infectious process in response to host resistance (16).

Hsps with a molecular mass of approximately 60 kDa (Hsp60) are some of the most predominant bacterial proteins, accumulating to high levels in stressed bacteria (7, 28). Although predominantly located in the cytoplasm of bacteria, there are reports suggesting that, in at least some pathogens, Hsp60 can be surface located and can mediate adherence to host cells and therefore play a role in virulence (11, 13). The abundance of this protein combined with its surface expression make it a major antigen, and its highly conserved nature could make it a common antigen providing some degree of cross-protection between different infections (17).

Numerous studies have indicated that Hsp60 from pathogens as diverse as Yersinia enterocolitica, Plasmodium yoelii, Histoplasma capsulatum, Helicobacter pylori, Mycobacterium tuberculosis, and Mycobacterium leprae is immunogenic. Immunization of animals with autologous Hsp60 can induce an immune response that is able to protect animals against a subsequent challenge (15, 21, 22, 24, 27, 29). For example, large numbers of Hsp60-specific CD4⁺ T cells have been identified in mice immunized with killed M. tuberculosis (18). Immunization with mycobacterial Hsp60 transfected into antigen-presenting cells has been shown to induce proliferation of CD8⁺ T cells, providing protection against disease (29). Clones of these activated T cells can be passively transferred, indicating that protection against disease is T-cell mediated (30). In the case of the Y. enterocolitica Hsp60, the degree of protection afforded after immunization was markedly enhanced by the use of interleukin-12 (IL-12) as an adjuvant, possibly because this promoted the induction of T-cell responses to Hsp60 (21).

Francisella tularensis is a facultative intracellular gram-negative bacterial pathogen that causes the disease tularemia. There are several subspecies, which differ in the severity of disease they cause in humans. Strains of F. tularensis subsp. tularensis are most virulent, with an infectious dose of less than 10 CFU and a mortality of 5 to 6% in untreated cases of cutaneous disease (6). Strains of F. tularensis subsp. holarctica cause a milder form of disease, and the mortality rate associated with cutaneous disease in humans is <0.5% (6). Both F. tularensis subsp. holarctica and F. tularensis subsp. tularensis are virulent in mice by all routes, with a median lethal dose (MLD) of approximately 1 CFU (6). The live vaccine strain (LVS) of F. tularensis is derived from an F. tularensis subsp. holarctica strain and is attenuated in humans. In mice, F. tularensis LVS is virulent when given by the intraperitoneal (i.p.) route of challenge but not when given by the subcutaneous route (6). Until recently, F. tularensis LVS has been used for vaccination of humans, providing protective immunity associated with a strong, lasting T-cell response (6, 32). Killed vaccines, which cause only antibody production, offer no protection against high-virulence strains (4). No single protein or subunit appears immunodominant for all individuals, and none of those examined so far has demonstrated protection against tularemia despite being combined with potent adjuvants (32).

It has previously been shown that the expression of the F. tularensis Hsp60 is up-regulated under growth conditions that are proposed to mimic those inside macrophages (7). However, this has been difficult to demonstrate on cultures grown within macrophages (12). T cells from individuals primed by vaccination with F. tularensis LVS showed responses to Hsp60 (8). In this study, we have set out to determine whether immunization with Hsp60 isolated from F. tularensis is able to induce an immune response that provides protection against tularemia. In order to bias the immune system towards a cellular response, IL-12 was included as an adjuvant following promising results from vaccine studies against Mycobacterium avium (31) and Y. enterocolitica (21).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein preparation. Cells of F. tularensis LVS (NDBR lot 4) or a lipopolysaccharide (LPS) O-side chain-deficient variant of LVS (capsule-deficient [Cap^–] LVS) (26) were grown on blood cysteine glucose agar (BCGA: 3-g/liter Lab-Lemco agar, 20-g/liter bacterial peptone, 5-g/liter NaCl, 12.5-g/liter Davis agar [pH adjusted to 8], 0.02% [wt/vol] cysteine, 0.02% [wt/vol] histidine, 0.5% [wt/vol] glucose, 50 ml of defibrinated bovine blood) for 72 h at 37°C. Cells were harvested into phosphate-buffered saline (PBS), washed, and heat killed at 65°C for 1 h. The bacteria were boiled in sample buffer (Bio-Rad, Hertfordshire, United Kingdom), and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Hsp60 was identified by Western blotting with a monoclonal antibody to GroEL (Sigma-Aldrich, Poole, United Kingdom). Hsp60 was excised from large-format unstained gels and electroeluted into 4x Laemmli buffer (0.1 M Tris-HCl [pH 7.3], 0.768 M glycine, 0.4% [wt/vol] SDS) by using the Hoefer gel eluter (Amersham Pharmacia, Buckinghamshire, United Kingdom) at 70 V for 2 h, following the manufacturer's instructions. The eluted product was pooled and purified by dilution (more than 20x) in ammonium bicarbonate (3.95 g/liter) and SDS (1 g/liter) and centrifuged over a dialysis membrane (10,000-molecular-weight cutoff; Vivascience, Lincoln, United Kingdom). The protein was then diluted (20x) in sterile water and centrifuged further, until significant concentration was achieved. Excess SDS was removed by cold precipitation (4°C) followed by microcentrifugation. Sections of gel above 60 kDa, known not to contain protein bands, were excised, eluted, purified, and concentrated in a similar manner. This was used to provide samples for control group immunizations. Protein was assayed by using the bicinchoninic acid protein assay (Pierce, Rockford, Ill.) with bovine serum albumin as a standard (Sigma). The identity of the eluted protein was confirmed by SDS-PAGE and Western blotting with the anti-GroEL antibody (Sigma) and visualized by enhanced chemiluminescence detection with the ECL system (Pharmacia).

Immunization of mice. Groups of BALB/c mice 6 to 8 weeks old (Charles River International, Margate, United Kingdom) were immunized i.p. on days 0 and 14 with 25 µg of Hsp60/100 µl of saline with or without 0.5 µg of murine IL-12. Control groups were immunized with either IL-12 only or 100 µl of saline containing eluted protein-free gel. IL-12 was kindly provided by Roche Pharmaceuticals. Blood samples were obtained from each mouse by tail vein bleeding immediately prior to challenge (day 28). The serum was separated by microcentrifugation and stored at –20°C until analysis. Groups of mice were either challenged or culled to provide spleen cells a further 14 days later.

Challenge with F. tularensis. F. tularensis LVS, F. tularensis subsp. holarctica HN63, and F. tularensis subsp. tularensis Schu S4 were used for challenges. The strains were harvested into PBS after growth on BCGA for 24 h at 37°C and diluted to a given optical density. Confirmation of the dose was achieved by plating out a serial dilution. Groups of mice were challenged on day 28 with either 10³ or 10⁴ MLD of LVS i.p. or 100 MLD of HN63 or 10 MLD of Schu4 subcutaneously as separate challenges. Mice were monitored for 14 days, and survival to a humane end point was recorded.

ELISA method: antibody production. For the enzyme-linked immunosorbent assay (ELISA), briefly, Immunlon II (Dynex Technologies, Chantilly, Va.) plates were coated overnight with a suspension of heat-killed LVS (protein concentration, 8 µg/ml) or 10 µg of purified LPS per ml in carbonate coating buffer (Sigma). Plates were washed and blocked with 5% Blotto in PBS-Tween (Bio-Rad, Hemel Hempstead, United Kingdom). One hundred microliters of serum was double diluted across the plate, starting at a 1/50 dilution, and incubated at 37°C for 1 h. A control serum and positive isotype control (Oxford Biotech Ltd., Oxford, United Kingdom) were also run. After washing, 100 µl/well of a 1/15,000 dilution of the appropriate antibody conjugated to horseradish peroxidase (Oxford Biotech, Ltd.) was added and the plate was further incubated for 1 h. After washing again, TMB substrate (3,3',5,5'tetramethylbenzidine) (Sigma) was added at 100 µl/well at room temperature, the reaction was stopped after 15 min by the addition of 2 M H₂SO₄ at 50 µl/well, and the absorbance was measured at 450 nm. The isotype control was used to provide a standard curve, which was then used to convert a mid-absorbance value for the sera to an equivalent number of nanograms per milliliter of the control antibody. Statistical significance between groups was calculated by using the unpaired two-tailed Student's t test.

Separation of T and B cells from mixed spleen populations. Single-cell preparations were incubated for 15 min at 4°C with magnetic microbeads conjugated to antimouse CD45R (B220) antibodies (Miltenyi Biotec) for positive selection of B cells. They were then washed in separation buffer (1% fetal calf serum and 2 mM EDTA in PBS) and passed through a MACS separation column (Miltenyi Biotec) attached to a magnet. B cells were magnetically retained. The column effluent was then incubated with microbeads conjugated to CD5 (Ly-1) antibodies (Miltenyi Biotec) for the positive selection of T cells and then passed through another column. The efficiency of this separation was examined by fluorescence-activated cell sorter (FACS), which found that the selected cells contained <5% contaminating T or B cells (data not shown).

T-cell macrophage assay. The T-cell macrophage assay was developed from a macrophage assay described by Bosio and Elkins (3) where growth of intracellular LVS is controlled by primed T cells. The mouse macrophage cell line, J774 (ECACC), was seeded at 5 x 10⁵/ml/well into 24-well plates (Corning) and incubated overnight to form a confluent layer of approximately 10⁶ cells. Cells were grown in L-15 medium with 10% fetal calf serum (both from Gibco) at 37°C in air. An overnight culture of LVS was diluted into PBS to a known optical density. A dilution of this (made in L-15) was overlaid on the J774 cells to give a multiplicity of infection of 10 CFU per macrophage in a total well volume of 200 µl for 30 min. Extracellular LVS cells were then killed by replacing the medium with L-15 with 10 µg of gentamicin per ml (Sigma) for 30 min. The monolayer was then washed and overlaid with either naïve or vaccinated whole-spleen mixed cells at a ratio of 1 spleen cell to 2 macrophages. Macrophages were also overlaid with purified T or B cells from vaccinated mice, at a ratio of 1 cell to 4 macrophages. The wells were incubated in L-15 with 2-µg/ml gentamicin at 37°C for 66 h. The cells were then gently washed and lysed, serially diluted, and plated onto BCGA to provide a bacterial count. Each count was an average of six duplicate wells, with five spleens included in each group. These counts were compared to a negative control of infected macrophages with no additives and a positive control of infected macrophages with 50-ng/ml gamma interferon (IFN-) (R&D Systems, Minneapolis, Minn.). The tissue culture supernatants were stored frozen for later cytokine analysis by using a FACS.

Cytokine measurements. Tissue culture supernatants were assayed for the presence of range of cytokines indicative of T- or B-cell responses. This was performed by using a cytometric bead array (BD Biosciences, Oxford, United Kingdom) in which the levels of tumor necrosis factor alpha, IFN-, IL-5, IL-4, and IL-2 can be measured by fluorescent antibody tagging followed by FACS analysis. The kit was used in accordance with the manufacturer's instructions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of Hsp60. Whole-cell lysates of F. tularensis LVS were separated by SDS-PAGE, and Hsp60 was eluted, purified, and concentrated. When subsequently analyzed by SDS-PAGE, this protein was demonstrated to migrate as a single band with a molecular mass of approximately 60 kDa (Fig. 1, lane A) The identity of this protein band was confirmed as Hsp60 by Western blotting with an anti-Hsp60 antibody (lanes E to H). When the purified protein was analyzed for the presence of LPS by using the Limulus amoebocyte lysate assay, the level of LPS was found to be below the detection limit of the assay. A control sample was prepared by subjecting a section of the gel, which did not contain any protein bands, to the same elution, purification, and concentration procedure outlined above for Hsp60.

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FIG. 1. Silver-stained gel (lanes A to D) and Western blot (lanes E to H) of F. tularensis Hsp60 and cell lysates. The Western blot was probed with anti-Hsp60 antibody. Lanes: A, purified Hsp60; B, lysate of F. tularensis LVS; C, lysate of F. tularensis HN63; D; Sigma wide-range molecular mass markers (kilodaltons); E; lysate of F. tularensis LVS; F; lysate of F. tularensis HN63; G, ECL molecular mass markers (kilodaltons); and H, purified Hsp60.

Antibody responses after immunization with Hsp60. Purified Hsp60 with or without the addition of the adjuvant IL-12 was first used to immunize groups of mice. Serum was taken 2 weeks postvaccination and measured for antibody to F. tularensis cells (Table 1). Two separate experiments with Hsp60 isolated on two separate occasions and adjuvanted with IL-12 were carried out. Antibody to F. tularensis cells was not detected in the control mice. There was some variation in the serum antibody titers between mice, within groups. All groups had high levels of immunoglobulin G1 (IgG1) antibody, but the levels of IgG2a were higher in mice that had received Hsp60 and IL-12 (P = 0.008, Student's t test). IgM titers were not detected in any of the immunized mice.

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TABLE 1. Antibody responses of groups of immunized mice immediately prior to challengea

The serum antibody response to purified LPS was also measured. IgG1 or IgG2a antibodies directed against LPS were not detected in any of the immunized or control mice. However, low levels of IgG3 and IgM to LPS were detected in sera from control mice, which was due to the presence of LPS in the protein-free gel used to prepare the control vaccinations. There was no difference in the levels of these antibodies between the controls and groups receiving Hsp60 vaccines (Table 1).

Cellular immune responses after immunization with Hsp60. The assay previously described by Bosio and Elkins (3) was used to investigate whether T-cell responses in immunized mice were able to control the replication of F. tularensis LVS in macrophages. Neither the presence of separated T cells nor mixed spleen cells from Hsp60-vaccinated mice demonstrated any ability to control an LVS infection in macrophages. However, primed spleen cells taken from mice that had recovered from sublethal infections were capable of limiting the replication (data not shown). The tissue culture medium from this assay was measured for cytokines. Although there was some increase in tumor necrosis factor alpha, there was no production of IFN- by the spleen cells from Hsp60-vaccinated mice.

Protection afforded by immunization with Hsp60. Hsp60-immunized mice were subsequently challenged by the i.p. route with F. tularensis strains of increasing virulence in humans (LVS, HN63, or Schu S4). The results (Table 2) indicated that the degree of protection to challenge was dependent on the virulence of the challenge strains and also whether IL-12 was included as an adjuvant.

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TABLE 2. Protection of mice from F. tularensis challenge after immunization with Hsp60

Animals that had been immunized with Hsp60 alone were only poorly protected against a subsequent challenge with any strain of F. tularensis. In contrast, animals that had been immunized with a mixture of Hsp60 and IL-12 developed an immune response that provided complete protection against 1,000 MLD of F. tularensis LVS and some protection against 100 MLD of F. tularensis HN63. Mice that had been challenged with 10 MLD of F. tularensis Schu S4 showed an increase in time to death (P = 0.0007).

Protection afforded by immunization with LPS-free Hsp60. Hsp60 isolated from a capsule-negative variant of F. tularensis LVS only provided limited protection against LVS challenge, even when adjuvanted with IL-12.

Immunization with LPS from F. tularensis is known to provide protection against a subsequent challenge with some strains of F. tularensis. Although we had not been able to detect LPS in purified Hsp60 by using the Limulus amoebocyte lysate assay, it is known that F. tularensis LPS reacts poorly in this assay (25). The presence of traces of LPS contaminating the purified Hsp60 was visualized by a Western blot probed with a monoclonal anti-LPS antibody (Fig. 2). The antibody was produced in house and is known to bind to the O-side chain of F. tularensis LPS, but not to other closely related species such as Francisella novicida (data not shown).

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FIG. 2. Western blot of Hsp60 isolated from F. tularensis LVS and cell lysates of F. tularensis LVS and of F. tularensis Cap– LVS mutant. Lanes A to C: A; purified Hsp60 from F. tularensis LVS; B, lysate of F. tularensis LVS; C; lysate of F. tularensis Cap– LVS mutant. Lanes A to C were all probed with anti-LPS antibody. Lanes D to G: D; lysate of F. tularensis LVS; E, lysate of F. tularensis Cap– LVS; F, purified F. tularensis LVS Hsp60, probed with anti-HSP60 antibody; G; ECL molecular mass markers (kilodaltons).

To eliminate the possibility that this LPS was contributing to protection, we isolated Hsp60 from a variant form of F. tularensis LVS (Cap^– LVS), which does not produce an O-antigen side chain (26). The purified Hsp60 migrated as a single band when analyzed by SDS-PAGE. Western blotting showed the presence of LPS in the Hsp60 eluted from LVS and the lack of LPS in the Cap^– variant of LVS (Fig. 2).

Mice that had been immunized with Hsp60 and IL-12 from the Cap^– LVS variant developed high levels of IgG1 antibody to heat-killed F. tularensis cells, comparable to levels generated by Hsp60 from LVS (Table 1). Sera from these immunized mice contained low levels of IgG2a and no detectable level of IgG3 or IgM to heat-killed F. tularensis cells. Mice vaccinated with the Cap^– LVS Hsp60 and IL-12 were not protected against challenge with F. tularensis HN63 (Table 2).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The findings that Hsp60 from a range of pathogens is able to induce an immune response that provides protection against disease prompted us to investigate whether Hsp60 from F. tularensis could provide protection against experimental tularemia.

We purified Hsp60 as a single protein from a bacterial lysate of F. tularensis LVS. Immunization with this protein provided protection against an otherwise lethal LVS infection in mice. The effectiveness of the vaccine was greatly enhanced by the inclusion of IL-12, which may have acted as a Th1 adjuvant. IL-12 has been shown to be pivotal for generation of Th1 response and has been used as an adjuvant, enhancing the effect of vaccination, for several intracellular pathogens, including M. tuberculosis (9), Listeria monocytogenes (33), and Y. enterocolitica (21). However, we were unable to demonstrate cell-mediated immunity in terms of recall T-cell response to infected macrophages, nor did we detect significant levels of secreted IFN- from these T cells. Immunization with Hsp60 adjuvanted with IL-12 provided some protection against a more virulent strain of F. tularensis, HN63. However, it failed to protect mice from disease after challenge with the highly virulent F. tularensis strain SchuS4 but did provide an increase in the time to death.

This pattern of protection against strains of increasing virulence was similar to the pattern of protection against the same strains after immunization with purified LPS from F. tularensis (10). We therefore considered that the protection afforded by Hsp60 might be due to contaminating LPS. We had been unable to detect LPS by using the standard Limulus amoebocyte lysate assay, which was not surprising, as it has been previously been reported that F. tularensis LPS has significantly reduced bioactivity (25). However, we were able to demonstrate the presence of contaminating LPS in the purified Hsp60 by Western blotting. To further investigate this possibility, we isolated Hsp60 from a variant strain of F. tularensis that does not produce the LPS O-antigen (26). Immunization of mice with this Hsp60 preparation provided significantly less protection from tularemia than vaccination with the LPS-Hsp60 preparation did.

These findings clearly suggest that the protection we had observed with Hsp60 was due at least in part to LPS, which was complexed or copurified with the protein. However, its degree of involvement in the induction of protection was not clear. Control mice showed no protection despite having been immunized with a protein-free preparation, which also contained low levels of LPS. Also, the survival of the vaccinated groups appeared to be linked to the levels of IgG2a directed against the whole organism rather than towards LPS. Other studies reporting protection against tularemia after LPS vaccination noted the generation of high levels of antibody directed against LPS, in particular IgG2a (5) or IgM (10). However our inability to demonstrate an active role for the primed T cells suggests that the protection seen after immunization is more consistent with a role for antibody to LPS rather than a role for an immune response to Hsp60.

Studies of other bacterial pathogens have noted that the protection afforded by immunization with isolated Hsps is not due solely to the known immunogen. In the case of M. tuberculosis, it has been shown that Hsp10 (GroES homolog) isolated from M. tuberculosis was able to induce an immune response that protected mice from a subsequent challenge but recombinant Hsp10, produced in Escherichia coli, was nonprotective. Further, the incubation of recombinant Hsp10 with a crude M. tuberculosis cell lysate and its subsequent purification restored the protective efficacy of Hsp10, which suggested it bound immunologically active molecules (23).

This finding is also in accordance with a study in which guinea pigs which had been immunized with purified recombinant Hsp60 or Hsp70 from M. tuberculosis were not protected from disease (34), whereas mice immunized with native Hsp60 were protected (18, 29).

In some cases then, it is clear that the cellular components that copurify with Hsps are responsible for the observed protection and that the Hsps may be acting as carrier molecules. Hsp60 has been repeatedly found contaminating LPS purifications of Pseudomonas aeruginosa, which has been explained as the result of a physical association. It is suggested that this LPS-Hsp60 mix forms the complex described as the common protective antigen (orginal endotoxin protein), which has successfully been used for vaccination against P. aeruginosa (14).

Other studies have reported that either entire Hsps or fragments of these proteins that contain T-cell epitopes can be artificially complexed with antigens enhancing the immune response. Thus T-independent antigens such as capsular polysaccharides and LPS can induce a limited T-cell response (1, 2, 10).

It is possible that the effects we have seen were not solely due to the presence of LPS in our preparations of Hsp60 but were also due to the immunostimulating effect of Hsp60. Hsp60 has been shown to stimulate macrophages via CD14, sharing the pathway with endotoxic LPS and activating the innate immune system (19). Vaccination with autologous Hsp60 conjugated to capsular polysaccharide of Streptococcus pneumoniae provided T-cell help to produce a response to otherwise T-cell-independent antigens and therefore provided a protective immune response (20). This effect was explained as Hsp60 directly activating the macrophages prolonging the stimulation of the immune system in response to the vaccine.

Overall, our findings add to the body of evidence indicating that the protection afforded by immunization with Hsp60 from pathogenic bacteria might not be directly due to the immunogenicity of this protein. Together these findings suggest that Hsps merit further attention as important modulators of the immune response to other antigens from pathogenic bacteria.

 
* Corresponding author. Mailing address: Bldg. 245, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wilts SP4 0NS, United Kingdom. Phone: 44 1980 613462. Fax: 44 1980 613284. E-mail: mghartley{at}dstl.gov.uk.

Editor: V. J. DiRita

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, July 2004, p. 4109-4113, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4109-4113.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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