Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming
© Gonçalves et al; licensee BioMed Central Ltd. 2007
Received: 22 March 2007
Accepted: 22 August 2007
Published: 22 August 2007
Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.
The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.
Tuberculosis (TB) remains a leading cause of infectious disease mortality worldwide, accounting for nearly 2 million deaths annually. Despite the availability of effective anti-TB therapy, the world's case burden of TB continues to climb, in part owing to the concurrent acquired immune deficiency syndrome pandemic. The widespread use of the current TB vaccine, M. bovis bacillus Calmette-Guérin (BCG), has failed to curtail the TB epidemic. Therefore, TB eradication will require the development of an improved vaccine, which, in turn, will require application of state-of-the-art vaccine technology and new strategies.
A new vaccine against TB would need to induce protection superior to that elicited by the BCG vaccine and to permit administration to healthy individuals, infected individuals and perhaps even individuals presenting the active form of the disease. Thus, various strategies have been employed for the development and evaluation of new TB vaccines. Recombinant BCG strains, DNA-based vaccines, live attenuated Mycobacterium tuberculosis vaccines and subunit vaccines formulated with novel adjuvants have shown promise in preclinical animal models . The ability of DNA vaccines to elicit Th1-biased CD4+ responses and strong cytotoxic T lymphocyte responses make them particularly attractive as weapons against M. tuberculosis infection.
Experimental data collected by our group over the last few years have shown that a DNA vaccine encoding the M. leprae 65-kDa heat shock protein (DNA-HSP65) has prophylactic and therapeutic effects in a murine model of TB [2–5]. The prophylactic effect initially obtained from this vaccine was equal to that elicited by BCG vaccine [3, 6]. However, we would like to optimize this DNA vaccine for use in humans, and the prime-boost strategy seems a very promising option.
Heterologous prime-boost strategy has shown promise in various models of pathogenic infections . The results have been highly encouraging both in augmenting and modulating vaccine-induced immunity. This strategy is based on the combination of live attenuated viruses or BCG with DNA vaccines or recombinant proteins . In experimental models of TB, the ability of prime-boost strategy to complement the protection provided by BCG vaccination has been assayed . Such studies have shown that DNA-prime that codifying M. tuberculosis genes (Apa, HSP65 and HSP70), BCG-booster induced a higher level of protection than BCG alone . However, boosting the BCG vaccine with a recombinant modified vaccinia virus Ankara (MVA) expressing M. tuberculosis 85A antigen also induced higher levels of antigen-specific CD4+ and CD8+ T cells and greater protection against aerosol challenge . Others have demonstrated that BCG-prime DNA-Rv3407 (M. tuberculosis 10 kDa protein)-booster induced a greater protection against TB than BCG alone . In the present study, we investigated the influence that the order and route of BCG vaccination in combination with DNA-HSP65 vaccine has on the induction of protective immunity against TB.
SPF female BALB/c mice, 6–8 weeks old, were purchased from the University of São Paulo – FMRP. All mice were kept under specific pathogen-free conditions in a BSL 3 facility. All animal studies were conducted in accordance with the Institutional Animal Care and Ethics Rules of University of São Paulo – Brazil.
The M. tuberculosis H37Rv (n° 27294; ATCC, Rockville, MD, USA) and M. bovis BCG (Pasteur strain) were grown in an incubator for 7 days at 37°C in 7H9 Middlebrook broth (Difco, USA) enriched with 0.2% (v/v) glycerol and 10% (v/v) OADC (Difco, USA) and was prepared as described .
The DNA vaccine pVAX-hsp65 (DNA-HSP65) was derived from the pVAX vector (Invitrogen, Carlsbad, CA, USA) and was constructed as described . Endotoxin levels were measured using the Limulus amebocyte lysate kit – QCL-1000 (BioWhittaker, Walkersville, MD, USA). Endotoxin levels for plasmid used in this study were ≤ 0.1 endotoxin units/μg of DNA.
Immunization and challenge infection
Heterologous prime-boost regimen combinations
3 doses of intramusculard
DNA-hsp65 – 15 days of interval
Blood collection and antibody evaluation
Prior to the first immunization (pre-immune serum) and 15 days after the last immunization, individual serum samples were colleted by retro-orbital sinus puncture. Antibody levels in samples were measured by enzyme-linked immunosorbent assay (ELISA) described .
Recombinant M. lepraehsp65
Clone pIL161, containing the DNA coding for the M. leprae HSP65, was transformed into electrocompetent DH5α Escherichia coli cells. Briefly, DH5α E. coli cells containing pIL161 were grown in the presence of ampicillin to an OD600 of 0.6. The expression of the recombinant protein was induced by the addition of IPTG (isopropril-thi-B-D-galactosídeo) 0.5 mM. The induced culture was incubated for another 4 h at 30°C and was harvested by centrifugation (5000 g, 5 min, 4°C), then the pellet was lysed by sonication at 60 Hz with two cycles of 60 s (Tomy-Seiko, Japan). After washed with 10 mL of CE buffer, the pellet was resuspended in 5 mL of UPE buffer and the suspension was gently shaken at room temperature for 15 min. The insoluble material was washed by centrifugation at 10000 g for 20 min, a 3.6 M ammonium sulfate stock solution was added followed by incubation on ice for 30 min. This fraction was dissolved in 50 mM phosphate buffer to produce the crude fraction. The recombinant M. leprae Hsp65 was first fractionated on a FPLC-GP-250 Plus system (MonoQ HR 5/5, Pharmacia Biotech) using 50 mM phosphate buffer and eluted with a 20–600 mM NaCl gradient under a flow rate of 1 mL/min. Subsequently, the protein solution (100 μg) was resolved on a HPLC system (Shimadzu Class VP) and recombinant M. leprae Hsp65 was collected and the homogeneity of the recombinant M. leprae Hsp65 preparations was analyzed by polyacrylamide gel electrophoresis. Protein concentrations and endotoxin levels were determined as previously described [5, 14].
The levels of IFN-γ, interleukin (IL)-12, IL-10, TNF-α, IL-4 and IL-5 in the spleen cell supernatants and in lung homogenates from immunized mice were measured by ELISA as previously described . The following capture antibody anti-mouse IFN-γ, IL-12, IL-10, TNF-α, IL-4 or IL-5 (R46A2, 15.6, JES5-2A4, mIL4R-M2 and TRFK5 clones, respectively; Pharmingen) were used. Cytokine-antibody complexes were detected by the addition of biotin anti-mouse IFN-γ, IL-12, IL-10, TNF-α, IL-4 or IL-5 (XMG1.2, C17.8, SXC-1, B11-3 and TRFK4 clones, respectively; PharMingen). Detection limits were 40 pg/mL (for IFN-γ and 10 pg/mL (for IL-12 and IL-10, TNF-α, IL-4 and IL-5).
The ELISPOT method was used to detect IFN-γ secretion by spleen cells from immunized mice. In brief, ELISPOT plates (BD Biosciences) were coated with capture IFN-γ antibody overnight at 4°C. After washed and blocked with complete medium, the plates were incubated for 2 h at room temperature. The spleen was removed from each mouse aseptically. Red blood cells were removed from the spleen cells preparations using red blood cell lysis buffer (NH4Cl 0,16 M/Tris 0,17 M/pH 7,65). Cells were placed in RPMI-C 1640 medium (R-6504 – Sigma, St. Louis, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% of fetal bovine serum (all from Gibco-BRL). The cells were incubated (2 × 106 cells/well) for 48 h at 37°C with 5% CO2, with medium, concanavalin-A (20 μg/well) or recombinant Hsp65 (10 μg/well) and then were discarded. Plates were washed with de-ionized water and PBS/Tween 20. Secondary biotinylated antibody was added for 2 h and incubated at room temperature, followed by washing with PBS/Tween 20. Streptavidin-alkaline phosphatase was added to the plates for 1 h, followed by washing and by the development of a colour reaction using the AEC substrate reagent kit (BD Biosciences). The reaction was stopped by rinsing the plate with running water. The spots were enumerated using an ELISPOT reader (Biosys – Germany).
Thirty days after challenge, aliquots of lungs harvested from infected, sham-immunized mice and from immunized, infected mice were incubated in digestion solution as described . Serial 10-fold dilutions were plated on supplemented 7H11 agar media (Difco, USA). Colonies were counted after 28 days of incubation at 37°C with 5% CO2, and the results were expressed as CFU (g/lung).
Preparation of lung cells
Lungs were washed with sterile PBS and were placed in Petri dishes containing incomplete RPMI-1640 (R-6504 – Sigma, St. Louis, USA). Then, they were fragmented and transferred to conical tubes containing 0.5 μg/mL of Liberase Blendzyme 2 (Roche, Indianapolis, IN, USA) in incomplete RPMI-1640. Samples were processed as previously described .
Fluorescence-activated cell sorter analysis
To evaluate T cell subsets, effector function and memory markers, the following mAbs and their respective isotype controls were used: anti-CD62L (clone MEL-14), anti-CD4 (clones H129.19 and RM4-5), anti-CD8 (clone 53-6.7), anti-CD44 (clone Ly-24); rat-IgG2a-fluorescein isothiocyanate, rat-IgG2a-phycoerythrin and rat-IgG2a-peridinin chlorophyll protein. All mAbs were purchased from Pharmingen and used according to the manufacturer instructions. Lymphocytes were analyzed by flow cytometry using the CellQuest software FACSort (Becton Dickinson, San Jose, CA). Ten thousand events per sample were collected, and three-color fluorescence-activated cell sorter analysis was performed. Expression of CD62Llo and CD44hi was performed by dot plot in CD4+ or CD8+ gated lymphocyte populations.
Lung samples were fixed in 10% buffered formalin. Five-micrometer sections were stained with hematoxylin-eosin and the granulomatous lesions were analyzed by light microscopy (Leica, Germany).
All data were analyzed individually and the values were expressed as mean ± SEM. When the values indicated the presence of a significant difference by analysis of variance (ANOVA), a Tukey-Kramer multiple comparisons test was used. Values of P <0.05 were considered significant.
DNA-HSP65 boosting of BCGin provides greater protection than other immunization strategies
BCGin/DNA induces enhanced humoral immune response
BCGin/DNA stimulates a Th1 immune response
To evaluate the specific cytokine production from spleen cell cultures of non-immunized and immunized-mice 15 days after the last immunization, the ELISA assay was performed. After specific stimulation with rHsp65, the spleen cells of all immunized-mice, BCGsc (2275 ± 807 pg/mL), BCGin (3256 ± 120 pg/mL), DNA-HSP65 (3139 ± 383 pg/mL), BCGsc/DNA (2931 ± 430 pg/mL), produced significant levels of IFN-γ in relation to PBS group (Fig. 2B). However, the spleen cells of BCGin/DNA immunized-mice produced significantly higher levels of detectable IFN-γ (4411 ± 799 pg/mL) compared with the levels provided by other immunized mice (Fig. 2B). A similar pattern of cytokine production was observed in relation to IL-12, with the exception of BCGsc group that did not produce significant levels of IL-12 in relation to PBS group (Fig. 2C). The other groups of immunized-mice produced different levels of IL-12: BCGin/DNA (5025 ± 747 pg/mL), BCGsc (2208 ± 1055 pg/mL), BCGin (3803 ± 385 pg/mL), DNA-HSP65 (2962 ± 474 pg/mL) and BCGsc/DNA (3806 ± 942 pg/mL) (Fig. 2C). On top of that, the levels of IL-12 produced by BCGin/DNA group were significantly higher than those produced by DNA-HSP65 group (Fig. 2C). In addition to identifying the cytokines IFN-γ and IL-12, which are associated with the Th1 pattern, we found that the BCGin/DNA immunization schedule stimulated significantly higher IL-10 production (627 ± 174 pg/mL) compared with that provided by BCGsc (237 ± 110 pg/mL) and BCGsc/DNA (393 ± 102 pg/mL) groups (Fig. 2D). Similar levels of IL-10 were produced by BCGin/DNA, BCGin and DNA-HSP65 groups. Besides, we verified that DNA-HSP65 (38 ± 7 pg/mL), BCGin/DNA (59 ± 10 pg/mL) and BCGsc/DNA (59 ± 6 pg/mL) immunized-mice also produced significant levels of IL-4 compared with PBS group (Fig. 2E). In relation to ELISPOT assay, significant number of IFN-γ producing cells was mainly observed in BCGin/DNA mice (45 spots) in comparison with the number detected in PBS group and in the other immunized groups: BCGin, DNA-HSP65, BCGsc/DNA and BCGin/DNA (Fig. 2F).
Maintenance of Th1-type response after challenge with M. tuberculosis
Cytokine production in lung homogenates after 30 and 70 days of challenge
time after challenge
immunized with (below) and infected with H37Rv
692 ± 155
1069 ± 170
890 ± 87
1436 ± 185◆, ●
1332 ± 138
2343 ± 397◆, *
634 ± 110
795 ± 98
866 ± 87
1473 ± 120◆, ●
906 ± 64
1753 ± 190◆, *
606 ± 111
1281 ± 146
1378 ± 213
1990 ± 310◆, ●
1624 ± 254
3476 ± 375◆, *
447 ± 131
926 ± 56
1090 ± 153
1562 ± 180◆, ●
1192 ± 186
2709 ± 322◆, *
42 ± 9
104 ± 18
430 ± 23◆
625 ± 79◆, ●
558 ± 25◆
684 ± 104◆, *
30 ± 9
84 ± 4
391 ± 58◆
407 ± 33◆
334 ± 14◆
572 ± 46◆, *
178 ± 41
328 ± 25
505 ± 39◆
506 ± 58◆
470 ± 45◆
662 ± 65◆, *
181 ± 43
388 ± 25
530 ± 22◆
574 ± 44◆
523 ± 58◆
710 ± 52◆, *
BCGin/DNA induces up-regulation of CD44hi/CD62Lloexpression in pulmonary T lymphocytes
CD4+/CD8+ cell numbers and expression of CD44hiCD62Llo or CD44lo/CD62Lhi in the lungs of mice
time after challenge
immunized with (below) and infected with H37Rv
2.4 ± 0,6
1 ± 0,1
5 ± 0,8
1,6 ± 0,2
9 ± 0,7◆
1,7 ± 0,6
10,2 ± 0,5◆
2,18 ± 0,7
10,2 ± 0,8◆
1,64 ± 0,1
12,8 ± 1,5◆, *
2,8 ± 0,5◆, *
1,4 ± 0,2
2,5 ± 0,06
4,5 ± 0,4◆
5,9 ± 0,6◆
4,7 ± 1,5◆
6,2 ± 1,5◆
0,6 ± 0,3
1 ± 0,3
1 ± 0,4
1,3 ± 0,3
1 ± 0,1
1,4 ± 0,2
CD4 + /CD44 hi /CD62L lo
6,1 ± 2
12,8 ± 2,6
10 ± 4,7
10,7 ± 2,5
11,8 ± 2
15,3 ± 4
1,5 ± 0,5
3,6 ± 0,9
4,5 ± 1,4
6 ± 1
3,4 ± 0,9
9 ± 2,2◆, *
CD8 + /CD44 hi /CD62L lo
7,2 ± 1,4
14,6 ± 3,1
14,8 ± 6,4
12,8 ± 4
14,5 ± 1,3
20,8 ± 0,6*
1 ± 0,3
2,5 ± 0,7
2 ± 0,8
2,2 ± 1,7
2,7 ± 0,3
5,2 ± 0,5◆, *
CD4 + CD44 lo /CD62L hi
14 ± 1,5
17 ± 1,42
19,5 ± 2,6◆
36,8 ± 5,4◆, ●
24,6 ± 2,5◆
35,6 ± 4,3◆, *
12 ± 2,4
22,2 ± 1,27
26,2 ± 7,5
37,5 ± 5◆, ●
30 ± 3,8
42,4 ± 5,4◆, *
CD8 + CD44 lo /CD62L hi
15,3 ± 2
19,38 ± 1,7
21,9 ± 4,1
29 ± 2,8◆
18,42 ± 5,6
41,2 ± 6,5◆, *
8,7 ± 2
16 ± 4,4
24,3 ± 7,1
22,6 ± 1,1◆, ●
27,45 ± 6,1
35,2 ± 7,3◆, *
Reduction of lung injury after BCGin/DNA vaccination
Thirty days after infection the lungs of BCGin immunized mice presented few granulomas with mild parenchyma injury. After 70 days of infection, this group was characterized by a more extensive inflammatory response (Fig. 5X). In the lungs of BCGsc mice, we observed sparse, well-defined granulomas, containing macrophages and surrounded by a few lymphocytic foci. These mice presented less parenchymal damage than did infected mice, but on day 70 after infection the granulomatous process was more intense than that observed on day 30 post-infection (Fig. 5C). In the BCGin/DNA group, the lung parenchyma presented less damage and smaller foci of mononuclear inflammatory infiltrates than in any other group. This infiltration was characterized by the presence of macrophages and few lymphocytes, as well as by rare granulomatous lesions. Similar characteristics were observed 70 days after infection (Fig. 5D). The lungs of DNA-HSP65 immunized mice presented compact granulomas with mild parenchyma damage after 30 days of infection. Conversely, on day 70 post-infection, an intense inflammatory reaction characterized by the presence of multiples granulomas and increased tecidual damage was observed (Fig. 5X). It is noteworthy that both groups receiving BCGin prime (BCGin/DNA, Fig. 5) presented less parenchymal injury than did those receiving BCGsc prime (Fig. 5X).
In this study, we showed that heterologous prime-boost vaccination using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads when compared with the other prime-boost schedules (data not shown) evaluated or three doses of DNA-HSP65 as a naked DNA. The DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCGin prime was also associated with better preservation of lung parenchyma. Our findings also suggest that the order of stimuli is more relevant to the modulation of immune responses after challenge than is the route of BCG administration. Despite the fact that BCGin/DNA immunization clearly induced greater protection than did BCGsc/DNA immunization, both stimulated similar levels of IFN-γ production.
Distinct prime-boost vaccination protocols have been evaluated in experimental TB models. Goonetilleke et al. reported that parenteral or intranasal BCG immunization induced comparable levels of antigen-specific CD4+ responses in the spleen . However, only intranasal BCG (BCGin) elicited specific T cell responses in the lungs. We demonstrated that, although parenteral and intranasal prime induced comparable IFN-γ levels at the site of the infection, the latter clearly decreased the bacterial load on the order of 3 log10, in relation to non-immunized, infected mice, and did not provoke lung injury when the challenge was performed 15 days after immunization schedules. A difference of 1,2 LOG10 between the same groups was verified when the challenge was performed 60 days after vaccination. In a similar prime-boost strategy, Mollenkopf et al. showed that a DNA vaccine improved the efficacy of intravenous BCG prime . Likewise, our results reinforce the hypothesis that a DNA booster can increase BCG immunogenicity. Notably, in our study, a single booster with DNA-HSP65 conferred considerable protection. Two main aspects merit emphasis. The first is that intranasal route employed in our study is less invasive, primes the lymphoid tissues (in the nasal and bronchial mucosa) and is easily applied in humans . The second is that, in addition to increasing BCG-related protection, prime-boost immunization also makes it possible to optimize DNA-HSP65 immunization. In a classical protocol of DNA vaccination, we employed a schedule of 3 or 4 doses at 15-day intervals. In previous studies, the protective efficacy of DNA vaccine has been demonstrated [2, 4, 5, 17]. Nevertheless, other authors have found that administration of a DNA vaccine provokes a pronounced, disorganized granulomatous response that leads to consolidation of lung tissues, and that there was no evident protection, whether the vaccine was used prophylactically or therapeutically . In an attempt to increase the protective effect and to minimize possible side effects of DNA-HSP65 vaccine, we included the BCGin/DNA prime-boost strategy in our study. This strategy exceeded our expectations when the perspectives described above were attended by a single DNA administration.
To understand the possible mechanisms involved in the up-modulation of the immune response, we sought and found a correlation between IFN-γ and IL-10 levels, as well as between IFN-γ levels and CFU numbers. Measuring IFN-γ production by antigen-specific T cells provides the best available immunological correlate of protection against TB . Although this immunological parameter of protection is currently in question , the results described here show that levels of "ex vivo" IFN-γ are closely associated with protection. Surprisingly, in the lungs of sham-immunized, infected mice and BCGin/DNA immunized mice, we found a positive correlation between IFN-γ and IL-10 levels after challenge and, as expected, a negative correlation between IFN-γ levels and CFU counts. In the lungs of BCGin/DNA mice, IFN-γ levels were approximately four times higher than those of IL-10, although IL-10 production was higher than in the lungs of sham-immunized, infected mice. There is little consensus in the literature regarding the role of IL-10 in mycobacterial infection. Absence of IL-10 in the early phase of infection favors increased resistance to mycobacteria . However, in IL-10 transgenic murine model, the presence of excess IL-10 did not inhibit the T cell response to mycobacteria infection. Thus, IL-10, which was initially found to be an inhibitor of IFN-γ secretion, had little effect on IFN-γ production in this experimental model. In addition, the IL-10 secreted from activated T cells appears to have little influence on the overall patterns of cytokine secretion in response to mycobacterial infection . In a more recent study, Jung, 2003 demonstrated that there was no difference between wild-type and IL-10 knockout mice in their ability to deal with mycobacterial infection . It seems reasonable to assume that the IFN-γ-mediated protection observed in our study was associated with the decreased bacterial load and, consequently, control of the infection, whereas the IL-10-mediated protection was, to a great extent, due to an anti-inflammatory effect related to protection against tissue damage. Since the bacterial clearance is followed by tissue repair, the two events are not mutually exclusive. However, the anti-inflammatory effect cannot be exclusively attributed to IL-10. The BCGin and BCGsc mice, despite presenting IL-10 levels that were statistically lower than those detected in mice in the other prime-boost immunized groups, also presented less parenchymal damage than did the sham-immunized mice. In this context, it is possible that an effector function is performed by soluble mediators, such as transforming growth factor-β, or by cell-cell contact mediated by regulatory T cells, although this has yet to be investigated. It is also of note that TNF-α levels were comparable among the various experimental groups. Since one of the strongest correlates of TNF-α-mediated protection is its role in granuloma formation , we expected to find differences among the groups. Although the numbers of CD8+ cells in the lungs were significantly higher in BCGin/DNA immunized mice, TNF-α levels were comparable among the groups, despite variations in cell number and cell constitution. These data should motivate a search for differential cytokines, chemokines and adhesion receptors that might prove to be markers of disease progression.
We also analyzed the effector/memory phenotype of T lymphocytes in the lungs. It is well known that effector cells express CD62Llo and CD44hi and are characteristically short-lived . We have previously shown that expression of CD44hi on CD4+ and CD8+ cells is related to protection against TB [2, 6]. However, Kipnis et al. recently reported that the transference of spleen cells expressing a resting/naïve phenotype (CD62Lhi/CD44lo) but not effector cells (CD62Llo/CD44hi) protected the recipients after challenge with M. tuberculosis . The authors suggested that the emergence of T cell memory from the naive subset induces IFN-γ-mediated protection. We found an increase of CD4+ and CD8+ populations that express CD44hiCD62Llo in the lungs of BCGin/DNA, however we did not find striking differences in the CD44loCD62Lhi populations among the distinct groups of immunized, infected mice.
In conclusion, we found that a DNA-HSP65 booster increased BCG-mediated protection and that the order of stimulation was a relevant correlate for this protection. This makes the BCGin/DNA strategy attractive because it does not preclude childhood BCG vaccination. Increased protective efficacy induced by the DNA-HSP65 booster appeared to be attributable to increased numbers of CD4+ and CD8+ cells expressing the effector phenotype CD62Llo/CD44hi, as well as to higher IFN-γ, IL-10 and IL-12 levels.
intranasal administration of the BCG
subcutaneous administration of the BCG
colony forming unit(s)
DNA vaccine encoding the M. leprae 65-kDa heat shock protein
modified vaccinia virus Ankara
phosphate buffered saline
recombinant 65-kDa heat shock protein
tumor necrosis factor-alpha
We thank Ana Rocha from Department of Pathology for histological assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Instituto do Milênio.
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