67 results on '"Miyaji EN"'
Search Results
52. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.
- Author
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Ferreira DM, Miyaji EN, Oliveira MLS, Darrieux M, Arêas APM, Ho PL, and Leite LCC
- Subjects
- Animals, Antibodies, Bacterial blood, Female, Immunoglobulin G blood, Mice, Mice, Inbred BALB C, Pneumococcal Infections prevention & control, Pneumococcal Vaccines genetics, Bacterial Proteins genetics, Bacterial Proteins immunology, Pneumococcal Vaccines immunology, Recombinant Proteins administration & dosage, Recombinant Proteins immunology, Vaccines, DNA genetics, Vaccines, DNA immunology
- Abstract
Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.
- Published
- 2006
- Full Text
- View/download PDF
53. Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A.
- Author
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Oliveira ML, Arêas AP, Campos IB, Monedero V, Perez-Martínez G, Miyaji EN, Leite LC, Aires KA, and Lee Ho P
- Subjects
- Adhesins, Bacterial biosynthesis, Adhesins, Bacterial genetics, Administration, Intranasal, Amino Acid Sequence, Animals, Antibodies, Bacterial blood, Bacterial Adhesion genetics, Bacterial Proteins genetics, Female, Immunoglobulin A analysis, Immunoglobulin G blood, Lactobacillus genetics, Lactobacillus metabolism, Lipoproteins biosynthesis, Lipoproteins genetics, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Protein Sorting Signals genetics, Saliva immunology, Species Specificity, Adhesins, Bacterial immunology, Antibodies, Bacterial analysis, Bacterial Adhesion immunology, Lipoproteins immunology, Pneumococcal Infections immunology, Pneumococcal Infections prevention & control, Pneumococcal Vaccines administration & dosage, Respiratory Mucosa immunology, Streptococcus pneumoniae immunology, Vaccination, Vaccines, DNA administration & dosage
- Abstract
Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 10(9) CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.
- Published
- 2006
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54. DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae.
- Author
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Ferreira DM, Arêas AP, Darrieux M, Leite LC, and Miyaji EN
- Subjects
- Animals, Bacterial Proteins genetics, Bacterial Proteins immunology, Bacterial Proteins toxicity, Cell Line, Cricetinae, Humans, Mice, Mice, Inbred BALB C, Pneumococcal Vaccines genetics, Recombinant Proteins immunology, Streptococcus pneumoniae genetics, Streptococcus pneumoniae immunology, Streptolysins immunology, Streptolysins toxicity, Vaccines, Conjugate administration & dosage, Vaccines, Conjugate genetics, Antibodies, Bacterial blood, Pneumococcal Infections prevention & control, Pneumococcal Vaccines administration & dosage, Streptococcus pneumoniae pathogenicity, Streptolysins genetics, Vaccines, DNA administration & dosage
- Abstract
The 7-valent polysaccharide conjugate vaccine currently administered against Streptococcus pneumoniae has been shown to be highly effective in high risk-groups, but its use in developing countries will probably not be possible due to high costs. The use of conserved protein antigens using the genetic vaccination strategy is an interesting alternative for the development of a cost-effective vaccine. We have analyzed the potential of DNA vaccines expressing genetically detoxified derivatives of pneumolysin (pneumolysoids) against pneumococcal infections, and compared this with immunization using recombinant protein. The purified recombinant pneumolysoid with the highest residual cytolytic activity was able to confer partial protection against a lethal intraperitoneal challenge, with the induction of high antibody levels. Immunization with DNA vaccines expressing pneumolysoids, on the other hand, induced a significantly lower antibody response and no protection was observed.
- Published
- 2006
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55. Intradermal immunization of mice with cholera toxin B-pneumococcal surface protein A fusion protein is protective against intraperitoneal challenge with Streptococcus pneumoniae.
- Author
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Arêas AP, Oliveira ML, Miyaji EN, Leite LC, and Ho PL
- Subjects
- Administration, Cutaneous, Animals, Female, Immunization, Mice, Mice, Inbred BALB C, Pneumococcal Vaccines administration & dosage, Vaccines, Synthetic administration & dosage, Bacterial Proteins immunology, Cholera Toxin immunology, Pneumococcal Infections prevention & control, Pneumococcal Vaccines immunology, Recombinant Fusion Proteins immunology, Vaccines, Synthetic immunology
- Published
- 2005
- Full Text
- View/download PDF
56. Expression and characterization of cholera toxin B-pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice.
- Author
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Arêas AP, Oliveira ML, Miyaji EN, Leite LC, Aires KA, Dias WO, and Ho PL
- Subjects
- Administration, Intranasal, Animals, Antigens, Bacterial genetics, Antigens, Bacterial pharmacology, B-Lymphocytes drug effects, Bacterial Proteins genetics, Cholera Toxin genetics, Escherichia coli genetics, Immunoglobulin A analysis, Immunoglobulin G analysis, Mice, Antibody Formation drug effects, B-Lymphocytes immunology, Bacterial Proteins pharmacology, Cholera Toxin pharmacology, Recombinant Fusion Proteins pharmacology
- Abstract
Cholera toxin B subunit (CTB) is responsible for CT holotoxin binding to the cell and has been described as a mucosal adjuvant for vaccines. In this work, the ctxB gene was genetically fused to the psaA gene from Streptococcus pneumoniae, a surface protein involved in its colonization in the host that is also considered a vaccine antigen candidate against this pathogen. The CTB-PsaA fusion protein was expressed in Escherichia coli, and the purified protein was used for intranasal immunization experiments in Balb/C mice. CTB-PsaA was able to induce both systemic and mucosal antibodies evaluated in serum, saliva, and in nasal and bronchial wash samples, showing that CTB-PsaA is a promising molecule to be investigated as S. pneumoniae vaccine antigen candidate.
- Published
- 2004
- Full Text
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57. Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge.
- Author
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Varaldo PB, Leite LC, Dias WO, Miyaji EN, Torres FI, Gebara VC, Armôa GR, Campos AS, Matos DC, Winter N, Gicquel B, Vilar MM, McFadden J, Almeida MS, Tendler M, and McIntosh D
- Subjects
- Animals, Antibodies, Helminth blood, Carrier Proteins genetics, Carrier Proteins metabolism, Cattle, Cells, Cultured, Fatty Acid Transport Proteins, Female, Helminth Proteins genetics, Helminth Proteins metabolism, Interferon-gamma biosynthesis, Mice, Mice, Inbred BALB C, Monocytes, Mycobacterium fortuitum enzymology, Mycobacterium fortuitum genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Recombinant Fusion Proteins metabolism, Schistosoma mansoni growth & development, Schistosoma mansoni pathogenicity, Schistosomiasis mansoni parasitology, Vaccines, DNA administration & dosage, beta-Lactamases genetics, beta-Lactamases metabolism, Carrier Proteins immunology, Helminth Proteins immunology, Membrane Transport Proteins, Mycobacterium bovis genetics, Schistosoma mansoni immunology, Schistosomiasis mansoni prevention & control, Vaccines, DNA immunology
- Abstract
The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.
- Published
- 2004
- Full Text
- View/download PDF
58. Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C.
- Author
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Mazzantini RP, Miyaji EN, Dias WO, Sakauchi D, Nascimento AL, Raw I, Winter N, Gicquel B, Rappuoli R, and Leite LC
- Subjects
- Animals, Antibody Formation immunology, Bacterial Proteins genetics, Blotting, Western, Chlorocebus aethiops, Diphtheria Toxin antagonists & inhibitors, Diphtheria-Tetanus-Pertussis Vaccine immunology, Enzyme-Linked Immunosorbent Assay, Escherichia coli genetics, Genetic Vectors, Male, Mice, Mice, Inbred BALB C, Mycobacterium bovis growth & development, Neutralization Tests, Serotyping, Tetanus Toxin antagonists & inhibitors, Tetanus Toxin biosynthesis, Vaccines, Combined, Vaccines, Synthetic immunology, Vero Cells, Adjuvants, Immunologic, Antibodies, Bacterial immunology, BCG Vaccine immunology, Bacterial Proteins biosynthesis, Mycobacterium bovis genetics, Mycobacterium bovis immunology, Tetanus Toxin immunology
- Abstract
In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.
- Published
- 2004
- Full Text
- View/download PDF
59. Expression of Streptococcus pneumoniae antigens, PsaA (pneumococcal surface antigen A) and PspA (pneumococcal surface protein A) by Lactobacillus casei.
- Author
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Oliveira ML, Monedero V, Miyaji EN, Leite LC, Lee Ho P, and Pérez-Martínez G
- Subjects
- Adhesins, Bacterial, Bacterial Proteins genetics, Carrier Proteins genetics, Gene Expression, Lacticaseibacillus casei genetics, Lipoproteins genetics, Lipoproteins immunology, Pneumococcal Vaccines genetics, Pneumococcal Vaccines immunology, Pneumococcal Vaccines pharmacology, Recombinant Proteins metabolism, Streptococcus pneumoniae genetics, Bacterial Proteins metabolism, Carrier Proteins metabolism, Lacticaseibacillus casei metabolism, Lipoproteins metabolism, Membrane Transport Proteins
- Abstract
A number of recent research works in lactic acid bacteria aim towards the design of new strains that could be used as live vectors for the delivery of antigens for oral vaccination, or other therapeutic molecules. In this work, an inducible expression system based on the Lactobacillus casei lactose operon promoter was used to express three important surface antigens of Streptococcus pneumoniae in this lactic acid bacterium: a virulence-related pneumococcal surface antigen (PsaA) and two variants of the virulence factor PspA (pneumococcal surface protein A). Expression of the three proteins was induced upon growth on lactose and strongly repressed by glucose. These proteins were produced intracellularly. Also, secretion to the growth medium was achieved by means of a fusion to the secreting and processing signals from the L. casei surface proteinase. Interestingly, while secreted PspA proteins were found in the culture supernatants, PsaA remained trapped in the cell wall. Expression of pneumococcal antigens in a food-grade organism opens an alternative for mucosal vaccination against this important pathogen.
- Published
- 2003
- Full Text
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60. Protective efficacy of PspA (pneumococcal surface protein A)-based DNA vaccines: contribution of both humoral and cellular immune responses.
- Author
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Miyaji EN, Dias WO, Tanizaki MM, and Leite LC
- Subjects
- Animals, Bacterial Proteins genetics, Female, Immunization, Mice, Mice, Inbred BALB C, Opsonin Proteins, Phagocytosis, Pneumococcal Vaccines administration & dosage, Streptococcus pneumoniae pathogenicity, Vaccines, DNA administration & dosage, Vaccines, DNA immunology, Antibodies, Bacterial blood, Bacterial Proteins immunology, Pneumococcal Infections prevention & control, Pneumococcal Vaccines immunology, Streptococcus pneumoniae immunology, Th1 Cells immunology
- Abstract
Streptococcus pneumoniae is a major public health problem and new strategies for the development of cost-effective alternative vaccines are important. The use of protein antigens such as PspA (pneumococcal surface protein A) is a promising approach to increase coverage at reduced costs. We have previously described the induction of a strong antibody response by a DNA vaccine expressing a C-terminal fragment of PspA. Fusion of this fragment with the cytoplasmic variant of SV40 large T-antigen (CT-Ag) caused reduction in specific interferon-gamma produced by stimulated spleen cells. In this work we show that the DNA vaccine expressing the C-terminal region of PspA elicits significant protection in mice against intraperitoneal challenge with a virulent strain of S. pneumoniae. Furthermore, fusion with CT-Ag completely abrogated the protection elicited by DNA immunization with this fragment. In this case, protection did not correlate with total anti-PspA antibody production nor with total IgG2a levels. The anti-PspA sera obtained from both constructs showed equivalent opsonic activity of pneumococci, indicating that the antibodies produced were functional. We could, though, observe a correlation between a lower IgG1:IgG2a ratio, which is indicative of a stronger bias towards Th1 responses, and protection. We also show that a vector expressing the most variable N-terminal alpha-helical region induces higher antibody formation, with increased protection of mice against intraperitoneal challenge with a more virulent strain of S. pneumoniae. As a whole, these results indicate that antibodies elicited against PspA would not be solely responsible for the protection induced by DNA vaccination and that cell-mediated immune responses could also be involved in protection against pneumococcal sepsis.
- Published
- 2003
- Full Text
- View/download PDF
61. Analysis of serum cross-reactivity and cross-protection elicited by immunization with DNA vaccines against Streptococcus pneumoniae expressing PspA fragments from different clades.
- Author
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Miyaji EN, Ferreira DM, Lopes AP, Brandileone MC, Dias WO, and Leite LC
- Subjects
- Animals, Antibodies, Bacterial blood, Antigens, Bacterial chemistry, Antigens, Bacterial genetics, Bacterial Proteins chemistry, Base Sequence, Cloning, Molecular, Cross Reactions, DNA, Bacterial genetics, Female, Immunization, Immunoglobulin G blood, Mice, Mice, Inbred BALB C, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments immunology, Pneumococcal Infections immunology, Pneumococcal Infections prevention & control, Bacterial Proteins genetics, Bacterial Proteins immunology, Pneumococcal Vaccines genetics, Pneumococcal Vaccines immunology, Streptococcus pneumoniae genetics, Streptococcus pneumoniae immunology, Vaccines, DNA genetics, Vaccines, DNA immunology
- Abstract
Streptococcus pneumoniae is a major cause of disease, especially in developing countries, and cost-effective alternatives to the currently licensed vaccines are needed. We constructed DNA vaccines based on pneumococcal surface protein A (PspA), an antigen shown to induce protection against pneumococcal bacteremia. PspA fragments can be divided into three families, which can be subdivided into six clades, on the basis of PspA amino acid sequence divergence (S. K. Hollingshead, R. Becker, and D. E. Briles, Infect. Immun. 68:5889-5900, 2000). Since most clinical isolates belong to family 1 or family 2, PspA fragments from members of both of these families were analyzed. Vectors encoding the complete N-terminal regions of PspAs elicited significant humoral responses, and cross-reactivity was mainly restricted to the same family. DNA vaccines encoding fusions between PspA fragments from family 1 and family 2 were also constructed and were able to broaden the cross-reactivity, with induction of antibodies that showed reactions with members of both families. At least for the pneumococcal strains tested, the cross-reactivity of antibodies was not reflected in cross-protection. Animals immunized with DNA vaccines expressing the complete N-terminal regions of PspA fragments were protected only against intraperitoneal challenge with a strain expressing PspA from the same clade.
- Published
- 2002
- Full Text
- View/download PDF
62. PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae.
- Author
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Miyaji EN, Dias WO, Gamberini M, Gebara VC, Schenkman RP, Wild J, Riedl P, Reimann J, Schirmbeck R, and Leite LC
- Subjects
- Adhesins, Bacterial, Animals, Antibodies, Bacterial biosynthesis, Bacterial Proteins genetics, Base Sequence, Carrier Proteins genetics, DNA, Bacterial genetics, Heat-Shock Proteins genetics, Humans, Immunity, Cellular, Interferon-gamma biosynthesis, Interleukin-4 biosynthesis, Lipoproteins genetics, Mice, Mice, Inbred BALB C, Pneumococcal Vaccines genetics, Pneumococcal Vaccines immunology, Streptococcus pneumoniae genetics, Th1 Cells immunology, Vaccines, DNA genetics, Vaccines, DNA immunology, Vaccines, DNA pharmacology, Bacterial Proteins immunology, Carrier Proteins immunology, Heat-Shock Proteins immunology, Lipoproteins immunology, Membrane Transport Proteins, Pneumococcal Vaccines pharmacology, Streptococcus pneumoniae immunology
- Abstract
Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.
- Published
- 2001
- Full Text
- View/download PDF
63. Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.
- Author
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Miyaji EN, Mazzantini RP, Dias WO, Nascimento AL, Marcovistz R, Matos DS, Raw I, Winter N, Gicquel B, Rappuoli R, and Leite LC
- Subjects
- Animals, Immunization, Male, Mice, Mice, Inbred BALB C, Antibodies, Bacterial biosynthesis, BCG Vaccine immunology, Bacterial Proteins immunology, Diphtheria Toxin immunology, Vaccines, Synthetic immunology
- Abstract
BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.
- Published
- 2001
- Full Text
- View/download PDF
64. Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice.
- Author
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Nascimento IP, Dias WO, Mazzantini RP, Miyaji EN, Gamberini M, Quintilio W, Gebara VC, Cardoso DF, Ho PL, Raw I, Winter N, Gicquel B, Rappuoli R, and Leite LC
- Subjects
- Animals, Antibodies, Bacterial biosynthesis, Brain microbiology, Interferon-gamma biosynthesis, Interleukin-4 biosynthesis, Male, Mice, Mice, Inbred BALB C, Mycobacterium bovis immunology, Whooping Cough prevention & control, Mycobacterium bovis genetics, Pertussis Toxin, Pertussis Vaccine immunology, Vaccines, Synthetic immunology, Virulence Factors, Bordetella immunology
- Abstract
The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.
- Published
- 2000
- Full Text
- View/download PDF
65. Photorepair prevents ultraviolet-induced apoptosis in human cells expressing the marsupial photolyase gene.
- Author
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Chiganças V, Miyaji EN, Muotri AR, de Fátima Jacysyn J, Amarante-Mendes GP, Yasui A, and Menck CF
- Subjects
- Animals, Cell Survival radiation effects, DNA radiation effects, DNA Fragmentation, Dose-Response Relationship, Radiation, Flow Cytometry, HeLa Cells, Humans, Microscopy, Fluorescence, Pyrimidine Dimers, Time Factors, Transfection, Apoptosis, DNA Repair, Deoxyribodipyrimidine Photo-Lyase genetics, Macropodidae genetics, Ultraviolet Rays
- Abstract
Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.
- Published
- 2000
66. Human Bcl-2 expression delays ultraviolet-induced apoptosis in marsupial cells.
- Author
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Miyaji EN and Menck CF
- Subjects
- Animals, Apoptosis physiology, Cell Line, Cell Survival radiation effects, Humans, Kinetics, Macropodidae, Proto-Oncogene Proteins c-bcl-2 genetics, Recombinant Proteins metabolism, Transfection, Apoptosis radiation effects, Proto-Oncogene Proteins c-bcl-2 metabolism, Ultraviolet Rays
- Abstract
We have introduced the human bcl-2 gene under the control of the human metallothionein MTIIA promoter into the rat kangaroo PtK2 cell line. Two independent clones were obtained in which the levels of Bcl-2 protein expression can be controlled by the addition of metals in the culture medium. These cell lines were employed to investigate the effects of this protein in UV-induced apoptosis. Overexpression of Bcl-2 in PtK2 cells resulted in a delay in the appearance of apoptosis markers, such as chromatin condensation and internucleosomal DNA fragmentation. However, colony survival after UV was not affected, suggesting that Bcl-2 did not impose a definitive block for cell death. The elimination of cyclobutane pyrimidine dimers through photoreactivation 24 h after irradiation in cells overexpressing Bcl-2 did not affect apoptosis. This indicates that irreversible events in the signaling pathway of apoptosis occur in the period between irradiation and photoreactivation even in the presence of high levels of Bcl-2 protein can delay the onset of UV-induced apoptosis in these marsupial cells, early events triggered by the pyrimidine dimers, upstream from the Bcl-2 action, lead the cell to a state committed to die.
- Published
- 1998
67. Ultraviolet-induced cell death is independent of DNA replication in rat kangaroo cells.
- Author
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Miyaji EN and Menck CF
- Subjects
- Animals, Aphidicolin pharmacology, Cell Death drug effects, Cell Line, DNA Repair, Darkness, Kidney, Kinetics, Macropodidae, Male, Time Factors, Cell Death radiation effects, DNA Replication drug effects, Ultraviolet Rays
- Abstract
Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained as for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication.
- Published
- 1995
- Full Text
- View/download PDF
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