Protective efficacy of different strategies employing Mycobacterium

Original Research

1.

Introduction

2.

Materials and methods

3.

Results

4.

Discussion

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis Patrícia RM Souza, Carlos R Zárate-Bladés, Juliana I Hori, Simone G Ramos, Deison S Lima, Tatiana Schneider, Rogério S Rosada, Lucimara GL Torre, Maria Helena A Santana, Izaíra T Brandão, Ana P Masson, Arlete AM Coelho-Castelo, Vânia L Bonato, Fabio CS Galetti, Eduardo D Gonçalves, Domingos A Botte, Jeanne BM Machado & Celio L Silva† †Universidade

de São Paulo, Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Paulo, Brasil

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drugresistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime–boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime–boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine. Keywords: heat-shock protein, liposome, prime-booster, tuberculosis, vaccine Expert Opin. Biol.Ther. (2008) 8(9):1255-1264

1.

Introduction

Mycobacterium tuberculosis infection remains a major global health emergency. More than eight million new cases of tuberculosis (TB) are detected annually and two million people die of this disease each year [1]. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastropically [2,3]. Despite the fact that most cases can be treated with antibiotics, this is long and needs the combination of at least three different drugs. Nonetheless, increasing rates of multi-drug resistant (MDR)-TB have been reported and recently a new kind of TB due to extensively drug-resistant (XDR) bacteria has been described [4]. Bacille Calmette-Guérin (BCG), the only available vaccine against TB, has become the most widely used vaccine throughout the world. It is estimated that 10.1517/14712590802324591 © 2008 Informa UK Ltd ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

more than 3 billion people have received it, but it is estimated to prevent only 5% of all potentially vaccinepreventable deaths due to TB. In a number of studies from different parts of the world, the protective efficacy of BCG against adult pulmonary TB has varied dramatically between 0 and 80% [5]. Thus, the development of a more effective vaccine to protect against TB is urgently needed. A broad array of reports testing different antigens and immunization strategies against tuberculosis have been published, including studies of DNA vaccines, protein- or peptide-based vaccines, recombinant BCG, and naturally or genetically attenuated strains of pathogenic mycobacteria [6]. However, all of these strategies have shown different protective patterns. The ability of DNA vaccines to elicit T helper type 1 (Th1)-biased CD4+ and strong cytotoxic T lymphocyte responses make them particularly attractive as new weapons against M. tuberculosis infection. This method of immunization is advantageous in terms of simplicity, adaptability and cost-effectiveness of vaccine production. On the other hand, in large animals and humans, DNA vaccines were not as immunogenic as expected, and the reasons for this lower efficacy are currently unknown [7]. In this regard, the development of effective strategies to deliver plasmid DNA designed to enhance the transfection of large animal cells is regarded as an important consideration for optimizing these vaccines [8]. We have demonstrated previously that intramuscular injection of naked DNA encoding the heat-shock protein 65 kDa gene from M. leprae (DNAhsp65) protects mice against subsequent challenge with virulent M. tuberculosis [9,10]. This DNA vaccine, which was initially designed to prevent tuberculosis, also showed pronounced therapeutic activity in mice infected previously [11-13]. Although naked DNAhsp65 elicits an effective immune response in this experimental model of tuberculosis, the amount of plasmid required is quite high. One reason for the high dose requirement is that extracellular delivery results in degradation of a considerable quantity of naked DNA by extracellular deoxyribonucleases [14]. Thus, novel methods for DNA delivery, including microspheres and liposomes, have presented promising results in different experimental models [15,16]. As such, these microparticles can be engineered to deliver immunostimulatory molecules to specific tissues and cells. Thus, they might also increase the biosafety of these molecules by limiting their distribution in vivo, thereby minimizing systemic toxicity, which is a major point to be considered in terms of DNA vaccination. Another advantage of these formulations is their biocompatibility and easy preparation, and several formulations are currently in clinical use [17]. In addition, the heterologous prime–boost strategy has been effectively tested in various models of pathogenic infections. In experimental models of tuberculosis, the ability of this strategy to complement the protection provided by BCG vaccination has been assayed [18,19]. This is of particular interest regarding the development of the much needed 1256

new tuberculosis vaccine, due to the ‘BCG primed’ status of entire populations in developing countries where this vaccine is routinely administered to newborns. Therefore, based on the need for alternatives to be designed to improve DNA and BCG immunizations, in the present study, we compared the protective responses of naked DNA, vectorized DNA and recombinant Hsp65 protein in liposomes and microspheres as well as a prime–boost strategy for DNAhsp65 vaccination in mice. We observed that all approaches tested elicited a protective immune response against tuberculosis, with remarkable advantages for liposome and prime–boost vaccinations. Thus, we conclude that based on the advantages of both systems, and since BCG vaccination is mandatory in endemic areas, the best strategy to consider for the development of an effective new TB vaccine is DNAhsp65 in liposomes. 2.

Materials and methods

2.1

Plasmid DNA production and quality control

The hsp65 gene from M. leprae was cloned into BamHI–NotI restriction sites of a pVAX1 vector (Invitrogen). The pVAX1-hsp65 construct (DNAhsp65) was prepared using an Endo-Free Plasmid Giga kit (Qiagen) following the manufacturer’s instructions. Cloned DNA was checked by restriction analysis and sequencing. The endo-free condition for DNA vaccination was determined by a Limulus amebocyte lysate (LAL) test as recommended by European and US Pharmacopeias [20] using the QCL 1000-LAL test kit (Cambrex). 2.2

Recombinant Hsp65 Protein (rHsp65)

Escherichia coli BL21 transformed with the plasmid peT28A encoding the M. leprae hsp65 gene were cultured in LB containing ampicillin (100 µg/µl). Bacterial growth was monitored by spectrophotometry at 600 nm in a Shimatzu UV-1650 spectrophotometer. When the OD reached a value of 0.6, the culture was induced with 0.1 M isopropylthiogalactoside (IPTG; Gibco, BRL) and incubated at 30°C under agitation for 4 h. rHsp65 purification was performed according to the protocol of Portaro et al. [21]. 2.3

Preparation of microspheres

Microspheres were obtained by the double emulsion/solvent evaporation technique. Briefly, 30 ml of dichoromethane solution containing 400 mg of poly DL-lactide-co-glycolide (PLGA) 50:50 or PLGA 85:15 (Resomer from Boehringer, Ingelheim) and 1.5 mg trehalose dimicolate (TDM, SigmaAldrich) were emulsified with 1.3 ml of an inner aqueous phase containing 5 mg of DNAhsp65 or 1 mg of rHsp65 protein using a T25 Ultraturrax homogenizer (IKA–Labortechnik) to produce a primary water–oil emulsion. This emulsion was then mixed with 100 ml of an external aqueous phase containing 1 – 3% poly(vinyl alcohol) (Mowiol 40 – 88, Aldrich Chemicals) as surfactant to form

Expert Opin. Biol.Ther. (2008) 8(9)

Souza, Zárate-Bladés, Hori, Ramos, Lima, Schneider, Rosada, Torre, Santana, Brandão, Masson, Coelho-Castelo, Bonato, Galetti, Gonçalves, Botte, Machado & Silva

a stable water-in-oil emulsion. The mixture was stirred for 6 h with a RW 20 IKA homogenizer for solvent evaporation. Microspheres were colleted and washed three times with sterile water, freeze-dried and stored at 4°C. 2.4

Liposome preparation

Lipids [Egg phosphatidylcholine (EPC), 1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl3-trimethylammonium-propane (DOTAP)] were purchased from Avanti Polar Lipids. The cationic liposomes were prepared in three steps: i) preparation of liposomes according to Bangham’s method [22]; ii) dehydration by lyophilization; and iii) hydration to obtain dehydrated–hydrated vesicles (DRVs) described by Kirby and Gregoriadis, [23]. Briefly, the desired amounts of all lipid stock solutions in chloroform (EPC/DOPE/DOTAP 50:25:25% molar) were mixed and dried to a thin film using a rotatory evaporator in a 650 mmHg vacuum for 1 h. The dried lipid film was hydrated with water at 30oC above its phase transition temperature. The liposomes were extruded through polycarbonate membranes (100 nm nominal diameter) 15 times at a nitrogen pressure of 12 kgf/cm2. Preformed empty dehydrated-hydrated liposomes were mixed with DNA in a NaCl final concentration equal to a saline solution (0.9%) at a temperature of 4oC and vortexed for size control. The cationic lipid:DNA molar charge ratio was 10. 2.5

Immunization protocols

Specific-pathogen-free female Balb/c mice, 6 – 8 weeks of age, were separated into six experimental groups with six animals in each group which received: i) saline; ii) BCG; iii) naked DNAhsp65; iv) liposomes containing DNAhsp65; v) microspheres containing DNAhsp65 or rHsp65; and vi) prime-boost with BCG and DNAhsp65, respectively. Immunizations were performed according to one of the following treatments (Figure 1): The BCG group received 105 BCG Morreau bacilli administered subcutaneously (s.c.). For naked DNAhsp65 vaccination, each mouse received 100 µg of DNA intramuscularly (i.m.) in four doses. For liposomes, a single dose with 25 µg of DNAhsp65 was given intranasally. For microspheres immunization, mice received two distinct formulations administered i.m. in a single dose. These microsphere formulations consisted of DNAhsp65 (30 µg) in TDM-loaded PLGA 50:50 microspheres, and rHsp65 (1.6 µg) protein in TDM-loaded PLGA 85:15 microspheres. Finally, for prime–boost vaccination, mice were primed with 105 BCG Morreau bacilli s.c. and boosted with 100 µg DNAhsp65 i.m. 2.6

Antibody evaluation

Serum from vaccinated mice was collected by orbital bleeding one day prior to challenge. To assess antigen-specific antibody levels, 96-well plates (Maxisorp Nunc-Immuno plates) were coated with 0.1 ml of rHsp65 purified protein

(5 µg/ml) in coating solution (14.3mM Na2CO3, 10.3mM NaHCO3, 0.02% NaN3, pH 9.6), incubated at 4°C overnight, and then blocked with 1% bovine serum albumin in PBS for 60 min at 37°C. Serum samples were applied in serial 10-fold dilutions from a starting dilution of 1:10. Each serum sample represented pooled sera from three vaccinated or control mice. After incubating the plates for 2 h at 37°C, antimouse IgG (B7022; Sigma-Aldrich), and IgG1 and IgG2a biotin conjugates (A85 – 1 and R19 – 15, respectively; BD Biosciences Pharmingen) were added for detection of specific antibody. After washing, plates were incubated at room temperature for 30 min with a StreptAB kit (Dako). To detect bound antibody, the o-phenylene diamine (OPD) substrate (Sigma-Aldrich) was added; the reaction was stopped with the addition of 50 µl of a 16% solution of sulfuric acid. The optical density (OD) was measured at 490 nm. The antibody titer was defined as the highest dilution of serum that gave an OD of 0.5. 2.7

Mycobacterium tuberculosis challenge

The experimental model of pulmonary tuberculosis was described previously by Bonato et al. [12]. Briefly, mice were anesthetized with 2,2,2-tribromoethanol (Acros) by intraperitoneal administration. The trachea was exposed and 1 × 104 viable bacilli of the H37Rv M. tuberculosis (number 27294; ATCC) strain were inoculated. The incision was sutured with sterile silk. All procedures were performed in a level III bio-safety room facility at the Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. 2.8

Cytokine spot ELISA (ELISPOT) assays

Cells from the lung were obtained as described above and were assayed for the frequency of IFN-γ and IL-4 producing cells by spot ELISA (ELISPOT; BD Biosciences) according to the manufacturer’s guidelines. In brief, 96-well nitrocellulosebottomed plates (Millipore IIA) were coated with mAb specific for IFN-γ or IL-4 by overnight incubation at 4°C. We incubated 2 × 106 lung cells with concanavalin A (Con-A) as a standard control or with rHsp65. After 48 h of incubation, cells were washed off and IFN-γ and IL-4 bound to the nitrocellulose were detected with biotinylated rat anti-mouse IFN-γ and rat anti-mouse IL-4 antibodies followed by streptavidin–alkaline phosphatase and substrate. Spots were counted by light microscopy. The frequency of IFN-γ and IL-4-producing cells for each T cell concentration was calculated by averaging the number of spots for triplicate wells. 2.9

Colony forming unit (CFU) determination

Lung tissue was prepared as described previously [12]. Briefly, aliquots of lungs harvested from saline-injected and from immunized infected mice were isolated and placed on a Petri dish containing incomplete RMPI 1640 medium (Sigma-Aldrich). After fragmentation, the samples were transferred to conical tubes and incubated under agitation at


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37°C for 30 min in digestion solution containing Liberase Blendzyme 2 (Roche) 5 µg/ml. Serial dilutions were plated on 7H11 Mildebroock agar medium supplemented with 10% fetal calf serum. Colonies were counted after 28 days of incubation at 37°C. 2.10

*

*

Figure 1. Experimental design. This figure shows the experimental time points for immunizations, infection and sample collection. Asterisks indicate the days when mice received each vaccination with the different formulations. The routes and doses administered are as follows: The saline group received 50 µl of saline into each quadriceps intramuscularly (i.m.) in four doses. For BCG, mice received one dose of 1 × 105 bacilli subcutaneously. For naked DNAhsp65 vaccination, each mouse received 100 µg of DNA i.m. in four doses. For liposomes, a single dose of 25 µg of DNAhsp65 was given intranasally. For microsphere immunization, mice received two distinct formulations administered i.m. in a single dose. These microsphere formulations consisted of DNAhsp65 (30 µg) in trehalose dimicolate (TDM)-loaded poly DL-lactide-co-glycolide (PLGA) 50:50 microspheres, and rHsp65 (1.6 µg) protein in TDM-loaded PLGA 85:15 microspheres. Prime–boost vaccination, mice were primed with 105 BCG Morreau bacilli s.c. and boosted with 100 µg DNAhsp65 i.m.

2.11

IgG 1

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Figure 2. Detection of HSP65-specific antibodies after immunization. IgG1- (gray bars) and IgG2a-specific (black bars) antibodies were detected in serum collected 15 days after the last immunization. The non-immunized group received saline solution. Bars represent the mean ± standard deviation. Asterisks indicate statistically significant differences (p < 0.05) compared to nonimmunized groups. Data are representative of three independent experiments with six mice per group. *IgG1. ‡IgG2a.

1258

Statistical analysis

All values are expressed as mean ± SEM. Data were investigated by analysis of variance (ANOVA) using the Bonferroni multiple comparisons test. Values of p < 0.05 were considered significant. 3.

4

Histology and morphometric analysis

To perform histological and morphometric analysis of lung parenchyma, the right lung was fixed with Carnoy’s solution and embedded in paraffin. Slices 4 µm thick were obtained using a microtome and stained with hematoxylin and eosin. Morphometric analysis was performed with an integrating eyepiece with a coherent system made of a 100-point grid consisting of 50 lines of known length, coupled to a conventional light microscope (Axioplan, Zeiss). A volume fraction of collapsed and normal pulmonary areas was determined by the point-counting technique at a magnification of × 400 across 10 random non-coincident microscopic fields. Points falling in tissue areas were counted and divided by the total number of points in each microscopic field. Thus, data were reported as the fractional area of pulmonary tissue [24].

Results

3.1 A mixed pattern of antibodies is raised by Hsp65 antigen

The humoral immune response elicited by immunization with the different strategies was determined by ELISA in serum collected one day prior to challenge. Figure 2 shows that the highest antibody titers specific for M. leprae Hsp65 antigen were produced in naked-DNAhsp65immunized mice. The microsphere and prime-boost groups were able to enhance the production of antibodies, as well. These three strategies elicited a mixed pattern of antibodies due to the production of both IgG1 and IgG2a, almost to the same levels in each group. In contrast, in liposome-immunized mice, neither of the IgG isotypes were detected, probably due to the intranasal route used for immunization. 3.2 Immunization with DNAhsp65 by different strategies induces and enhances a predominantly T helper type 1 (Th1) immune response during active TB

In previous studies, we have shown a predominance of the Th1 immune response after four doses of naked DNAhsp65 vaccination [25,26]. In the present study, we compared immune responses elicited by different immunization strategies using naked DNA and lower doses of DNAhsp65 vectorized


Souza, Zárate-Bladés, Hori, Ramos, Lima, Schneider, Rosada, Torre, Santana, Brandão, Masson, Coelho-Castelo, Bonato, Galetti, Gonçalves, Botte, Machado & Silva Medium IL-4 IFN-γ

ELISPOT (spots/106 cells)

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Figure 3. Frequency of IL-4- (open bars) and IFN-g-producing (filled bars) T lymphocytes in lungs. Mice were immunized using the different strategies and infected 15 days after the last immunization. IFN-γ- and IL-4-producing T cells, specific for rHsp65, were estimated by spot elisa (ELISPOT) 30 days after challenge. Bars represent the mean ± standard deviation. Asterisks indicate statistically significant differences (p < 0.05). Data are representative of three independent experiments with six mice per group. The control of the experiment is shown in the inset figure where the production of the cytokines was stimulated with Con-A or culture medium. *Relative to the saline group. ‡Relative to the DNAhsp65 group.

in microspheres/liposomes with or without rHsp65, and by a prime-boost strategy with BCG and DNA. The animals vaccinated with these strategies and infected with virulent M. tuberculosis showed increased numbers of IFN-γ producing T cells in response to rHsp65 protein when compared with the saline group. In addition, a marked reduction of IL-4-producing T cell clones was detected only in mice immunized with liposomes when compared with the saline group (Figure 3). These results indicate that Hsp65 immunization primed a Th1 immune response either by intranasal, or intramuscular routes. In addition, the reduction of DNAhsp65 dose is also possible when using liposomes or microspheres as vaccine delivery systems. 3.3 DNAhsp65 with different delivery systems and prime-boost strategy protects mice against M. tuberculosis infection

All strategies elicited substantial protective immunity against virulent M. tuberculosis as shown in Figure 4. Infection was

done 15 days after completion of immunizations and bacterial counts in the lungs were determined four weeks later. We observed an approximately 1.5 log reduction in bacterial counts in the lungs of all immunized animals. No difference was detected between the different vaccination strategies (Figure 4). Furthermore, histological analysis was performed on the lungs of infected mice. We observed that in the saline group, the lung parenchyma showed intense inflammation. The affected area presented few infiltrating lymphocytes and a high number of foamy macrophages. In contrast, mice that received immunization with naked DNAhsp65, liposomes (DNAhsp65), microspheres (DNAhsp65/rHsp65), and the prime-booster (BCG/DNAhsp65) strategy showed a lower pneumonic area with a cell infiltrate containing a predominance of lymphocytes and macrophages (Figure 5). Moreover, the histological evaluation was confirmed by a morphometric analysis, which determined that ∼ 70% of the lung was damaged in the non-vaccinated group, in


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Log10 (CFU/lung)

Saline DNAhsp65 Prime-boost Microspheres

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Figure 4. Bacterial numbers in lungs of immunized mice. M. tuberculosis H37Rv colony forming units (CFU) counts were performed 30 days after challenge. The total amount of DNAhsp65 administered in each strategy is shown for all groups on the x-axis. Bars represent the mean ± standard deviation. Values of p < 0.05 were considered significant. Data are representative of three independent experiments with six mice per group.

contrast to all mice immunized with DNAhsp65 by different methods (Figure 6). Taken together, these data showed the efficacy of Hsp65 in priming a pro-inflammatory immune response necessary to kill M. tuberculosis and that this response is well controlled in vaccinated mice avoiding the damage to lung parenchyma as occurs in non-vaccinated animals. 4.

Discussion

The increased number of tuberculosis cases and the appearance of new, extremely resistant strains of M. tuberculosis (XDR-TB) reveal the alarming status of this disease worldwide [27]. Despite considerable scientific efforts directed at developing a new antituberculosis vaccine, BCG remains the only available option. A specific vaccine designed to prevent tuberculosis or disease reactivation from the latent state is necessary and many proposals are in development, some of which are being tested in clinical trials [28]. Nonetheless, even in the event that some of these candidates are able to demonstrate good protection, it is likely that any activity will be variable among different populations, as is the case with BGC. In this sense, the optimization of novel vaccine candidates needs to continue. In recent years, we and others have shown that i.m. injection of mycobacterial Hsp65 antigen delivered as a DNA vaccine is able to protect mice against TB. Moreover, the DNAhsp65 vaccine can also be used as a therapeutic agent in active disease [11] or associated with current antituberculosis drugs resulting in a more rapid form of treatment for TB and is effective against MDR-TB, as well [25]. Although the success of naked DNAhsp65 in protecting against and treating TB 1260

has been shown, multiple doses and large amounts of plasmid DNA are required for effective immunization [29]. Our laboratory had directed efforts to optimize DNAhsp65 vaccination. An initial attempt was made by encapsulating DNAhsp65, with TDM as an adjuvant, into biodegradable microspheres of PLGA. A single dose of these microspheres containing only 30 µg of DNA conferred protection against experimental TB at the same level as 400 µg of naked DNA administered three times [15]. Another promising strategy for vaccine optimization is the heterologous prime–boost immunization regimen. In this sense, PLGA microspheres with slow- and fast release characteristics were used to encapsulate DNA and rHsp65 protein, respectively, aimed at DNA priming and protein boosting. Results showed a higher and sustained production of IFN-γ-specific anti-Hsp65 antibodies when compared with DNA–immunized animals [30]. Moreover, immunization using the same mixture of microspheres was effective in a guinea-pig model as well [31]. In subsequent experiments, we tested intranasal BCG as a primer with an intramuscular booster of DNAhsp65 and again, high levels of mycobacterial diminution were achieved with preservation of lung parenchyma [18]. Finally, we recently developed a novel formulation of cationic liposomes [32] and used them as DNAhsp65 vaccine delivery systems. These lipid structures have the advantage of simplicity, low cost, and safety, and are widely employed in a great variety of products already on the market. Considering all of this, it was essential to determine if some of these strategies present additional benefits in the prophylaxis of TB. Thus, we tested this hypothesis comparing: i) four doses of naked DNAhsp65; ii) a single dose of liposome containing DNAhsp65; iii) prime–boost using microspheres containing DNAhsp65 and rHsp65 protein; and iv) prime–boost of BCG and DNAhsp65. Regarding the humoral immune response, the different strategies elicited a marked production of both IgG1 and IgG2a antibodies. The highest levels were observed in mice immunized with four doses of naked DNA. Nonetheless, a single dose of microspheres and BCG/DNA was able to elicit significant production of antirHsp65 antibodies. This result showed the effectiveness of microspheres in antigen delivery. Although we could not detect anti-IgG-specific antibodies in the liposome group of mice, this is probably due to the intranasal immunization procedure, where the IgA subtype is predominant [33]. The ability of DNAhsp65 to induce protective effects in murine and guinea-pig models of TB is attributed to the induction of cellular immune responses. This immunostimulation was associated with IFN-γ-producing CD4+ and cytotoxic CD8+ T cells [34]. Moreover, mycobacterial Hsp65 is able to act as a chaperone, probably facilitating antigen presentation and processing due its peptidase activity [21], stimulate dendritic cells via Toll-like receptor 4 (TLR-4), leading to the development of cytolytic T cell responses [35],


Souza, Zárate-Bladés, Hori, Ramos, Lima, Schneider, Rosada, Torre, Santana, Brandão, Masson, Coelho-Castelo, Bonato, Galetti, Gonçalves, Botte, Machado & Silva A.

B.

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Figure 5. Histological evaluation of lungs of immunized mice 4 weeks after challenge. Histological analyses of lungs from mice immunized with the different strategies and challenged with M. tuberculosis. (A) Non-infected; (B) Saline; (C) DNAhsp65; (D) Liposome; (E) Microspheres; (F) Prime-boost; (G) BCG. The photographs show the intense inflammatory process in the saline group in contrast to the observed in Hsp65-vaccinated animals and BCG group, where the inflammation was less severe and lung architecture was preserved.


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Figure 6. Morphometric analysis of lung parenchyma 30 days after M. tuberculosis challenge. Cellular infiltrates in lung parenchyma were measured and expressed as a percentage of the total area of lung tissue analyzed. The total amount of DNAhsp65 administered in each strategy is shown for all groups. Bars represent the mean ± standard deviation. Values of p < 0.05 were considered significant relative to the saline group. Data are representative of three independent experiments with six mice per group.

and enhancing cross-presentation by dendritic cells independently of TLR recognition [36]. Thus, we compared the cellular immune response elicited by the different strategies using a single dose of DNA. Our results demonstrated that all vaccinated groups showed higher production of IFN-γ by Hsp65-specific T cells after M. tuberculosis challenge. In contrast, levels of IL-4-producing T cells were lower. Interestingly, in liposome-immunized mice, the frequency of this last type of lymphocyte was even more reduced. These data were in accordance with the reduction of bacilli counts and preservation of lung parenchyma. At this point, essentially none of the strategies presented fundamental differences in the type of immune response elicited in our model. Thus, the obvious question is which one of these should be chosen for further evaluation and for clinical trials. We believe that several important points must be considered to answer this question. First, in the simplest manner, the safer formulation appears as the best option. In this regard, considering that all formulations are based in the use of a DNAhsp65 vaccine, this is a point of utmost importance considering some concerns in relation to the use of genetic vaccines. These include the possibility of integration into the cell genome, silencing or activating essential genes or oncogenes respectively, vertical transmission if DNA vaccine is taken up by germ cells, and the possibility of harmful cross-reactions against genomic DNA among others [37,38]. Nonetheless, several reports have not demonstrated these phenomena and in a recently study where DNAhsp65 vaccine was assessed for toxic effects in cancer patients, no adverse effects of relevance were presented in immunized individuals [39]. 1262

Second, the most cost-effective approach should be considered. In this regard, liposomes carrying DNA would be the best option. This approach combines efficiency, great ease in generation, and improvement in safety due to the enormous reduction in DNA used (16-fold less), and the avoidance of the necessity for attenuated bacilli as is the case for BCG, which can potentially become dangerous in some subjects, such as children with malnutrition. Nonetheless, one important aspect that liposomes could circumvent in clinical practice is the low efficacy in priming immunity of some DNA vaccines tested in humans, and although the reasons for these phenomena is unknown, it is probably related to the transfection efficiency [7]. Moreover, even though the vaccine currently available worldwide for TB, BCG, confers variable protection [40], there are some other aspects that must be taken into account regarding whether BCG should be replaced. One of these is the so-called ‘non-targeted’ effects of BCG in infants in low-income countries. Although the existence of these effects was recognized at the start of BCG immunizations more than 75 years ago, the real determination of ‘BCG co-protective’ effects were largely neglected and remains one of the poorly characterized phenomena in TB. Another important issue is that in the case of a new antigen demonstrating improvement in protection against TB, as could be the case for the current vaccine candidates being tested in clinical trials, what should be done about previously BCGvaccinated subjects? [41]. Further complicating this situation is the fact that although these trials have shown good results, it is not clear whether they can be directly extrapolated to the rest of the Third World populations considering the probabilities of genetic variations [42]. In addition, and as supported by others, BCG vaccination has presented many more advantages than disadvantages for these populations [41-43]. Therefore, we support the idea that BCG could not simply be replaced, at least not in the near future. In conclusion, considering the data and all factors regarding new anti tuberculosis vaccination strategies presented here, we believe that because BCG vaccination is mandatory in endemic areas, a second immunization with vectorized DNAhsp65 in liposomes could be the most realistic option for consideration for clinical evaluation. Finally, further studies designed to test the therapeutic activity of the formulations evaluated here are also needed.

Acknowledgements We are grateful to technical assistance.

Elaine

Medeiros

Floriano

for

Declaration of interest This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.


Souza, Zárate-Bladés, Hori, Ramos, Lima, Schneider, Rosada, Torre, Santana, Brandão, Masson, Coelho-Castelo, Bonato, Galetti, Gonçalves, Botte, Machado & Silva activation of CD8+ cells, interferon-γ recovery and reduction of lung injury. Immunology 2004;113(1):130-38

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Affiliation Patrícia RM Souza1, Carlos R Zárate-Bladés1, Juliana I Hori1, Simone G Ramos2, Deison S Lima1, Tatiana Schneider1, Rogério S Rosada1, Lucimara GL Torre3, Maria Helena A Santana3, Izaíra T Brandão1, Ana P Masson1, Arlete AM Coelho-Castelo1, Vânia L Bonato1, Fabio CS Galetti4, Eduardo D Gonçalves4, Domingos A Botte4, Jeanne BM Machado4 & Celio L Silva†1 †Author for correspondence 1Universidade de São Paulo, Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, FMRP-USP, 14049-900 Ribeirão Preto, SP, Brasil 2Departamento de Patologia, FMRP-USP, 14049-900 Ribeirão Preto, SP, Brasil 3Universidade de Campinas, Faculdade de Engenharia Química, UNICAMP, 13083-970/6066, SP, Brasil 4Farmacore Biotecnologia Ltda., Campus da USP-Ribeirão Preto, 14049-900 Ribeirão Preto, SP, Brasil Tel: +55 16 36023086; Fax: +55 16 36336840; E-mail: [email protected]