a Department of Respiratory Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan b Department of Bacteriology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan c Department of Biochemistry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan
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Abstract |
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Introduction |
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Recently, DNA vaccines against various infections such as influenza,9 tuberculosis,10,11 hepatitis B,12 malaria,13 herpes simplex virus type 214 and HIV15 have been studied. In these studies, it was demonstrated that direct injection of DNA expression vectors encoding viral or bacterial proteins produced protective antibody and elicited cell-mediated immune responses. Compared with orthodox vaccines containing bacterial or viral antigens, the merit of DNA vaccination is that one can evaluate whether the protein encoded by the transgene induces an immune response without the need for protein purification.
Because the mecA sequence is a unique genetic marker for MRSA and PBP9' is located on the outer surface of the cytoplasmic membrane where it may be recognized easily by the host immune system, we hypothesized that DNA vaccination with the mecA sequence might elicit protective immunity against MRSA.
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Materials and methods |
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The 2008 bp mecA sequence was amplified by PCR from chromosomal DNA from the MRSA isolate N315, and used as a template.2,7 Unique restriction sites for HindIII were added to both ends of the mecA coding sequence by primers as follows: 5'-CCCAAGCTTACCATGAAAAA-GATAAAAATTGTTCCAC-3' and 5'-CCCAAGCTTT-TATTCATCTATATCGTATTTTTTATT-3'. After digestion with HindIII, the mecA sequence was subcloned into the multiple cloning site of the mammalian cell expression plasmid pCMV.SV+ (kindly provided by Dr R. G. Crystal, The New York HospitalCornell University Medical College) driven by the cytomegalovirus major immediate/early promoter/enhancer.16 A plasmid with a right-oriented mecA sequence, designated pCMV.mecA(+) and a plasmid with the mecA sequence in the opposite orientation, pCMV.mecA(), which was used as negative control, were obtained. The nucleotide sequence of mecA in pCMV.mecA was determined by dye terminator cycle sequencing and was identical to the previously reported sequence.7
Bacterial isolates and culture
MRSA LR5P1-IPM8-1, a highly resistant isolate derived from N315, and clinical isolate MRSA 1191 were used.17 Western blotting with anti-PBP 2' polyclonal antibody (see below) confirmed the presence of PBP 2' in crude extracts of membranes from LR5P1-IPM8-1 and MRSA 1191. These MRSA isolates were grown in LB medium containing methicillin 5 µg/L (Sigma, St Louis, MO, USA) for 16 h at 37°C. As a control, PBP 2'-free MSSA 209P was used. 209P was grown in antibiotic-free LB medium. Following serial dilution and 16 h culture on 1.5% heart infusion agar (Eiken Chemical Co. Ltd, Tokyo, Japan), the number of bacteria was estimated by spectrophotometry and the number of cfu counted.
Polyclonal antibody against PBP 2' (anti-PBP 2' antibody)
Rabbit polyclonal anti-PBP 2' antibody (IgG fraction) was kindly provided by Dr M. Saito, Dainabot Co., Ltd Research Center, Japan.18 This antibody targets the 477500 amino acid sequence of PBP 2' near the C-terminus.
Preparation of semi-purified PBP 2'
Exponentially growing MRSA LR5P1-IPM8-1 was harvested, washed in calcium- and magnesium-free phosphate-buffered saline (PBS), pH 7.4, and resuspended at 30 mg dry wt/mL with PBS containing lysostaphin 20 mg/L (Sigma). The suspension was first incubated at 25°C for 5 min, then 10 mM MgCl2 and DNase I 30 mg/L (Sigma) were added and the mixture was incubated at 37°C for a further 5 min.19 The membrane fraction was collected by centrifugation at 40,000g for 20 min at 4°C, and resuspended in SDSpolyacrylamide gel electrophoresis (SDSPAGE) sample buffer.20 To obtain semi-purified PBP 2', the membrane fraction was electrophoresed and the gel strip containing a 76 kDa PBP 2' protein band (confirmed by concomitant Western blotting), was cut out and electroeluted from the gel. Semi-purified PBP 2' was dissolved in a solution consisting of 5 mM Tris and 25.8 mM glycine and stored at 4°C. Protein content was assayed by BCA protein assay reagent (Pierce Chemical Co., Rockford, IL, USA).
Immunization of mice
Plasmid pCMV.mecA(+) or pCMV.mecA() at 150 µg in 0.1 mL of sterile PBS was injected im in 5-week-old female BALB/c mice, purchased from Charles River Japan Inc. (Yokohama, Japan). Mice were allocated at random into the various treated groups. Injection of plasmid alternatively into either side of the quadriceps muscle was performed three times at 2-week intervals, at day 0 (starting day), day 14 and day 28. At day 42, blood was drawn by cardiac puncture after anaesthaesia. Individual sera were prepared by centrifugation and were heat inactivated at 56°C for 30 min. Aliquots of each serum were used for Western blotting and enzyme-linked immunosorbent assay (ELISA) to measure the amount of anti-PBP 2' antibody.
Anti-PBP 2' antibody production in DNA-vaccinated animals
Membrane fractions of MRSA (4.5 µg/mm slit) were applied to a 715% gradient polyacrylamide gel and transferred to a PVDF membrane (Immobilon-PSQ, Millipore, Bedford, MA, USA). The blots were blocked in Blockace (Dainippon Pharmaceutial Co. Ltd, Tokyo, Japan) containing 0.1 mg/mL bovine serum albumin (Fraction V, Sigma). Then, the blots were probed with 1:500 diluted sera obtained from mice vaccinated with either pCMV.mecA(+) or pCMV.mecA() or with anti-PBP 2' polyclonal antibody at 100 ng/mL as a positive control, for 2 h at 25°C. As a second antibody, 1:5000 diluted horseradish peroxidase-labelled goat anti-mouse Ig (IgM + IgG + IgA, H + L) (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) was used for mice sera and 1:5000 diluted horseradish peroxidase-labelled goat anti-rabbit IgG for control anti-PBP 2' antibody. The blots were incubated for 1 h at 37°C, and developed with the ECL Western blotting system (Amersham International Plc, Amersham, UK).
Titre of anti-PBP 2' antibody in sera of DNA-vaccinated animals
The concentration of anti-PBP 2' antibodies was determined by ELISA with the sera obtained from mice vaccinated with plasmid pCMV.mecA(+) or pCMV.mecA(). Semi-purified PBP 2' (100 µL at 5 mg/L) in PBS containing 0.05% Nonidet P-40 was applied to each well of a 96-well tissue culture plate (Primaria, Microtest III, Becton-Dickinson, Lincoln Park, NJ, USA) for 16 h at 4°C. The wells were then blocked with Blockace containing 0.1% BSA for 1 h at 25°C. Preliminary studies demonstrated that ELISA was optimal with 1:500 diluted sera (data not shown). Aliquots of individual sera (1:500 diluted with 10% Blockace in distilled water) were added in duplicate, and kept for 16 h at 4°C. As a negative control, serum obtained from age- and sex-matched sham PBS-injected BALB/c mice were used. To calculate antibody concentration, serially diluted rabbit polyclonal anti-PBP 2' antibody or normal rabbit IgG (Sigma) was used instead of mouse serum. A second antibody comprising a 1:5000 dilution of horseradish peroxidase-labelled goat anti-mouse Ig (IgM + IgG + IgA, H + L) or a 1:5000 dilution of horseradish peroxidase-labelled goat anti-rabbit IgG was used for mouse sera and rabbit antibody, respectively. After incubation with the second antibody for 1 h at 25°C, colorimetric analysis was performed with the TMB peroxidase E1A substrate kit (Bio-Rad Laboratories, Richmond, CA, USA) following the manufacturer's instructions, and the plates were read at 450 nm using a microplate reader (Bio-Rad).
Phagocytosis of MRSA by murine phagocytes
Resident peritoneal macrophages were harvested from naive 6-week-old female BALB/c mice by peritoneal lavage with cold sterile PBS. The cell viability was consistently greater than 98% by determination with trypan blue dye-exclusion and more than 95% of these cells were monocyte-macrophages. The cells were washed with sterile PBS and resuspended in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% calf serum (complete medium). The cells were plated in each chamber of an eight-well Lab-Tek chamber glass slide (Nunc Inc., Napierville, IL, USA) at a cell density of 8 x 104/well and incubated for 90 min at 37°C in a CO2 chamber. Non-adherent cells were removed by rinsing with complete medium. Adherent cells were then covered with 300 µL of the above medium containing MRSA LR5P1-IPM8-1 or MSSA 209P, and 5% heat-inactivated (56°C, 30 min) sera from the mice vaccinated with pCMV.mecA(+) or pCMV.mecA().21 The ratio of bacteria to macrophages was 50:1. After 1 h incubation in 5% CO2, the slides were washed with PBS, fixed in ethanol and stained with Diff-Quick. The number of bacteria within the cells was counted using a microscope at 600x magnification, and the phagocytic index was calculated as a product of the percentage of positive ingestion and the average number of ingested bacteria per cell.22
Experimental bacteraemia in DNA-vaccinated mice
To assess the antibacterial effect of DNA vaccination on the development of renal infection, we induced in-vivo bacteraemia in experimental mice. To determine the number of bacteria required to produce bacteraemia, 107 or 108 cfu of MRSA 1191 was injected via the tail vein in non-immunized 11-week-old female BALB/c mice (day 0). At 1, 2, 4, 6 or 8 days after bacterial inoculation, both kidneys were excised, rinsed in sterile PBS and weighed. A minimum of three animals were used at each time point. Weighed kidneys were homogenized in sterile PBS at a constant volume of 1 mL/200 mg kidney. The solution was mixed thoroughly and a 100 µL aliquot was inoculated on to a 10 cm heart infusion agar dish. The number of colonies was counted after 16 h. To evaluate the antibacterial effect of DNA vaccination against experimental bacteraemia, 5-week-old female mice were allocated at random into three groups, which received mecA-expressing plasmid, negative control plasmid or sham treatment before bacterial challenge. Each group consisted of 20 mice. Mice were injected with 150 µg of plasmid or PBS three times at 2-week intervals. At 14 days after the last injection, 108 cfu of MRSA 1191 was inoculated via the tail vein (day 0). The kidneys were excised and number of bacteria in kidneys was evaluated at day 8.
Detection of PBP 2' in the supernatant of MRSA culture
To investigate the presence of PBP 2' in the supernatant of MRSA cultures, two isolates of MRSA (LR5P1-IPM8-1 and 1911) and MSSA 209P were grown in LB medium for 16 h at 37°C. Two per cent (v/v) of bacteria were transferred into new LB medium and incubated at 37°C until the optical density at 660 nm reached 0.6. The incubated solution was further kept at 4°C. Four mL of culture was filtered through a 0.2 µm nylon membrane. One hundred microlitres of filtered solution was inoculated on heart infusion agar. The remaining solution was mixed with trichloroacetic acid (TCA) at 10% final concentration for 10 min at 4°C to precipitate the proteins. After centrifugation at 18,000g for 10 min at 4°C, the pellets were rinsed twice with acetone and then air dried. The pellets were then resuspended in SDSPAGE sample buffer and subjected to 7% SDSPAGE. Simultaneously, cell pellets from 1 mL of cultured solution were resuspended in SDSPAGE sample buffer and electrophoresed. Western blot analysis was performed with 100 ng/mL anti-PBP 2' polyclonal antibody, as described above.
Data analysis
All values are reported as means ± S.E.M. Data were analysed using the Statview 4.5 program (Abacus Concepts Inc., Berkeley, CA, USA). Comparison of data expressed as titres or phagocyte index was made by unpaired t-test. Data on experimental bacteraemia were analysed using ANOVA with a post hoc Scheffe's and BonferroniDunn test for pairwise comparisons. A probability of <0.05 was considered significant.
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Results |
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Animal immunization with mecA plasmid
There were no deaths among animals who received either plasmid pCMV.mecA(+) or pCMV.mecA(). Body weight gain was comparable among the groups treated with pCMV.mecA(+) and pCMV.mecA(). No induration was detectable at the site of injection at 0, 24 and 48 h following the injection of plasmid. We evaluated the presence of antibody in the sera from animals immunized with mecA-expressing plasmid by Western blotting. The membrane fraction of MRSA was blotted on to a PVDF membrane and 1:500 diluted sera obtained from the animals immunized with plasmid pCMV.mecA were used as a primary antibody. Sera from the pCMV.mecA(+)-treated mice contained an antibody that reacted with PBP 2' (Figure 1, lane 1), but sera from mice treated with pCMV.mecA() did not react with PBP 2' (Figure 1, lane 2).
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Using an ELISA, the concentration of antibodies against semi-purified PBP 2' was measured in sera from mice immunized with PCMV.mecA. The antibody titres were 131.3 ± 29 ng/mL equivalent of standard rabbit anti-PBP 2' antibody (n = 5) in the sera of mice treated with PCMV.mecA(+), calculated from standard rabbit anti-PBP 2' antibody (Figure 2). Sera from mice treated with negative control plasmid contained 11.3 ± 10.1 ng/mL equivalent (n = 5) (P < 0.05) (Figure 2).
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Phagocytosis of MRSA
Although the mice treated with pCMV.mecA(+) produced antibodies against PBP 2', this did not necessarily indicate an antibacterial effect against MRSA. To determine whether the anti-PBP 2' antibody was protective, we evaluated the phagocytosis of MRSA by peritoneal macrophages. Phagocytosis of MRSA in the presence of 5% sera obtained from the mice vaccinated with pCMV.mecA(+) showed a five-fold increase compared with that of the sera of mice treated with negative control plasmid (Figure 3, left panel). The phagocytic index of pCMV.mecA(+)-treated mice was 1.52 ± 0.12 (n = 4) and that of negative control plasmid pCMV.mecA() was 0.29 ± 0.02 (n = 4) (P < 0.001). In contrast, the MSSA phagocytic index of serum from pCMV.mecA(+)-treated mice was 0.45 ± 0.12 (n = 4) and that of serum from pCMV.mecA()-treated mice was 0.53 ± 0.08 (n = 4) (P > 0.5) (Figure 3, right panel). Importantly, sera from pCMV.mecA(+)-vaccinated animals did not enhance phagocytes of MSSA. Thus immunization with plasmid pCMV.mecA(+) generated antibodies against PBP 2'that would enhance Fc receptor-mediated phagocytosis of MRSA but not MSSA.
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Preliminary studies showed that the number of bacteria in the kidney after challenge with 108 cfu of MRSA became constant after 6 days (Figure 4a). To investigate the antibacterial effect of DNA vaccination, 108 cfu of MRSA were inoculated and the number of bacteria in the kidneys was examined at day 8. Animals surviving and thus available for sampling at day 8 comprised 18 mice vaccinated with mecA-expressing plasmid, 17 mice vaccinated with negative control plasmid, and nine mice that received sham treatment, respectively. The number of bacteria in the kidneys obtained from the mice vaccinated with pCMV.mecA(+) was 1.48 ± 0.27 x 105 cfu/mg kidney, and was significantly lower than that of mice vaccinated with negative control plasmid pCMV.mecA() (3.59 ± 0.57 x 105 cfu/mg kidney) (P < 0.02) or that of sham-treated mice (3.43 ± 0.66 x 105 cfu/mg kidney) (P < 0.02) (Figure 4b).
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PBP 2' in the supernatant of MRSA culture
Two isolates of MRSA and one of MSSA were cultured in vitro and, during exponential growth, the cultures were filtered through a 0.22 µm nylon membrane. No bacteria were grown after inoculation of each filtrate on 1.5% heart infusion agar plate. Western blot analysis revealed that the filtrates of the two isolates of MRSA contained PBP 2', but there was no PBP 2' band in filtered samples from the supernatant or from cell pellets of MSSA (Figure 5). Although we could not exclude the possibility of bacterial lysis during filtration, this experiment shows the possibility that PBP 2' was released outside the MRSA cell wall.
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Discussion |
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Various components of S. aureus such as teichoic acid and protein A are recognized as antigens, and the production of antibodies against the bacteria is induced in infected animals and patients.27 Many investigators have attempted to induce immunity against the bacteria as prophylaxis for S. aureus infection, and circulating antibody was elicited by live or dead S. aureus in animal experiments and clinical studies.28,29,30,31 Recently, it was revealed that immunity against a capsular polysaccharide of S. aureusprompted phagocytosis of bacteria in vitro and in animal models.32,33,34 These studies indicated that the host can produce functional antibodies against MSSA by component(s) of S. aureus and encouraged the development of immunization against MRSA by other approaches such as DNA vaccination. The merits of DNA vaccination using PBP 2' are that it is not necessary to purify the antigen, and that the immunity elicited by the mecA DNA vaccine is expected to be aimed specifically at MRSA, and not at MSSA.
We detected anti-PBP-9 antibody in the sera of animals vaccinated with mecA-expressing plasmids. These sera increased phagocytosis of MRSA but not MSSA by peritoneal macrophages, demonstrating that antibody induced by mecA DNA vaccine was functional against MRSA in vitro. Sera were used after heat inactivation so as to minimize the influence of nonspecific complement-mediated phagocytosis. In addition, an in-vivo antibacterial effect of anti-PBP-9 immunity elicited by the mecA-expressing plasmid was observed in experimental mouse bacteraemia with MRSA. Both in-vitro phagocytosis and in-vivo bacteraemia experiments indicated that anti-PBP-9 antibodies recognized the PBP-9 protein of bacteria. Our results raise the question of how antibody against PBP-9 could easily bind to PBP-9 inside the cells. Although the PBP-9 molecule is located at the outer surface of the cytoplasmic membrane, the cell wall (peptidoglycan and teichoic acid) and the capsule would be obstacles to this linkage. Hence, we hypothesized that PBP-9 might escape from the bacterial cytoplasmic membrane through the cell wall. In fact, PBP-9 was detectable in the supernatant of MRSA cultures. However, we cannot completely exclude contamination by fragments of bacteria released during this procedure, although we filtered the solution carefully without excess pressure.
Another problem in assessing anti-staphylococcal humoral immunity is protein A, which binds to Fc of IgG antibodies. In Western blot analysis with whole membrane fractions of MRSA, sera from DNA-vaccinated mice reacted with a single 76 kDa band corresponding to PBP 2' even in the presence of protein A. This suggests that anti-PBP 2' antibodies elicited by DNA vaccination are specific for the PBP 2' molecule. However, it remains unresolved whether anti-PBP 2' antibody could bind directly on the surface of MRSA bacteria. Use of Fab fragments of anti-PBP 2' antibody would provide one approach to studying the binding of the antibodies to bacteria. However, at present, it is hard to obtain sufficient anti-PBP 2' antibody for purification of Fab fragments by our protocol. If available, use of protein A-deleted or -inactivated MRSA will be another choice.
In studies of experimental bacteraemia, the mice that received negative control plasmid exhibited higher survival rates than PBS-treated mice after MRSA challenge; however, the number of bacteria in the kidneys of the mice treated with negative control plasmid were higher than those for mice treated with the mecA-expressing plasmid, and almost the same as the number of bacteria in the kidney of sham-treated mice. One explanation for the survival of mice treated with negative control plasmid might be nonspecific up-regulation of immunity by unmethylated nucleotide sequences of the DNA of pCMV.mecA() plasmid.35
Delayed hypersensitivity to cell-wall extracts of staphylococci has been reported in mice repeatedly infected with S. aureus.36 It was characterized by footpad swelling at 48 h accompanied by infiltration of mononuclear cells and could be transferred to non-infected recipients by T lymphocytes from infected animals. Recipients of immune T cells developed severe necrotic lesions at sites inoculated with staphylococci. From this finding, it is questionable whether T cell immunity against staphylococci is favourable for the host. As there is no obvious skin lesion at the sites of DNA vaccine injection in this study, the possibility of delayed hypersensitivity appears to be low. However, it is still unknown whether the cellular immunity that might be induced by DNA vaccination could contribute to the antibacterial effect against MRSA.
In the future, we plan to evaluate whether the mecA DNA vaccine prevents the morbidity and mortality associated with MRSA infection, by using various animal models manifesting sepsis, peritonitis or pneumonia. We will also address the effects of DNA vaccination on MRSA carriage.
In summary, we have shown that mecA DNA vaccination elicited production of antibodies against PBP 2' in immunized animals. The antibodies increased phagocytosis of MRSA but not MSSA by peritoneal macrophages. In addition, an antibacterial effect was demonstrated in vivo in DNA-vaccinated mice infected with MRSA. These results suggest that PBP 2' or the mecA sequence may be eligible as a candidate molecule for vaccination against MRSA.
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Acknowledgments |
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Notes |
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References |
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Received 23 December 1998; returned 8 April 1999; revised 30 June 1999; accepted 15 July 1999