RESUMO
Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.
Assuntos
Galactosilceramidas , Infecções Pneumocócicas , Streptococcus pneumoniae , Humanos , Animais , Camundongos , Lipossomos , Pós , Infecções Pneumocócicas/prevenção & controle , Proteínas de Bactérias , Imunização , Vacinas Pneumocócicas , Imunoglobulina G , Pulmão , Anticorpos Antibacterianos , Camundongos Endogâmicos BALB CRESUMO
PspA is an important pneumococcal vaccine candidate that is capable of inducing protection in different animal models. Because of its structural diversity, a PspA-based vaccine should contain at least one fragment from each of the two major families (1 and 2) in order to elicit broader protection. In the present work, we have tested the potential of PspA hybrids containing fused portions of family 1 and 2 (PspA1ABC-4B and PspA1ABC-3AB) PspA fragments to induce protection against pneumococci bearing distinct PspA fragments. Sera from mice immunized with these hybrid PspA fragments were able to increase C3 deposition on pneumococci bearing PspA fragments from both families, in contrast with sera made against the PspA family 1 (PspA1ABC) and PspA family 2 (PspA3ABC) fragments, which were effective only within the same family. Although PspA hybrids were able to extend protection against pneumococcal infection with strains bearing diverse PspA fragments, the immunity elicited by family 2 was clade dependent, suggesting that PspA fragments from family 2 clades 3 and 4 should both be included in a comprehensive PspA vaccine. These results indicate that PspA fusion proteins constitute an efficient immunization strategy for future PspA-based antipneumococcal vaccines since they are able to extend protection provided by a protein derived from a single transcript.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas do Sistema Complemento/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Proteínas de Bactérias/biossíntese , Feminino , Soros Imunes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologiaRESUMO
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.
Assuntos
Apoptose , Reparo do DNA , Desoxirribodipirimidina Fotoliase/genética , Macropodidae/genética , Raios Ultravioleta , Animais , Sobrevivência Celular/efeitos da radiação , DNA/efeitos da radiação , Fragmentação do DNA , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Células HeLa , Humanos , Microscopia de Fluorescência , Dímeros de Pirimidina , Fatores de Tempo , TransfecçãoRESUMO
Increased nasopharyngeal colonization density has been associated with pneumonia. We used experimental human pneumococcal carriage to investigate whether upper respiratory tract viral infection predisposes individuals to carriage. A total of 101 healthy subjects were screened for respiratory virus before pneumococcal intranasal challenge. Virus was associated with increased odds of colonization (75% virus positive became colonized vs. 46% virus-negative subjects; P=0.02). Nasal Factor H (FH) levels were increased in virus-positive subjects and were associated with increased colonization density. Using an in vitro epithelial model we explored the impact of increased mucosal FH in the context of coinfection. Epithelial inflammation and FH binding resulted in increased pneumococcal adherence to the epithelium. Binding was partially blocked by antibodies targeting the FH-binding protein Pneumococcal surface protein C (PspC). PspC epitope mapping revealed individuals lacked antibodies against the FH binding region. We propose that FH binding to PspC in vivo masks this binding site, enabling FH to facilitate pneumococcal/epithelial attachment during viral infection despite the presence of anti-PspC antibodies. We propose that a PspC-based vaccine lacking binding to FH could reduce pneumococcal colonization, and may have enhanced protection in those with underlying viral infection.
Assuntos
Proteínas de Bactérias/imunologia , Fator H do Complemento/imunologia , Imunidade Inata , Nasofaringe/imunologia , Infecções Pneumocócicas/imunologia , Infecções Respiratórias/imunologia , Viroses/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Anticorpos Antibacterianos/biossíntese , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Coinfecção , Fator H do Complemento/química , Fator H do Complemento/genética , Mapeamento de Epitopos , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade nas Mucosas , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Nasofaringe/microbiologia , Nasofaringe/virologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Infecções Pneumocócicas/virologia , Ligação Proteica , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Mucosa Respiratória/virologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidade , Viroses/patologia , Viroses/virologiaRESUMO
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.
Assuntos
Apoptose/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Raios Ultravioleta , Animais , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Humanos , Cinética , Macropodidae , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Morte Celular/efeitos da radiação , Replicação do DNA , Raios Ultravioleta , Animais , Afidicolina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Escuridão , Rim , Cinética , Macropodidae , Masculino , Fatores de TempoRESUMO
Aluminum salts have been widely used in vaccine formulations and, after their introduction more than 80 years ago, only few vaccine formulations using new adjuvants were developed in the last two decades. Recent advances in the understanding of how innate mechanisms influence the adaptive immunity opened up the possibility for the development of new adjuvants in a more rational design. The purpose of this review is to discuss the recent advances in this field regarding the attempts to determine the molecular basis and the general mechanisms underlying the development of new adjuvants, with particular emphasis on the activation of receptors of innate immune recognition. One can anticipate that the use of these novel adjuvants will also provide a window of opportunities for the development of new vaccines.
Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Vacinas/imunologia , Fatores de Virulência/imunologia , Adjuvantes Imunológicos/química , Compostos de Alumínio/imunologia , Animais , Humanos , Imunidade Celular/imunologia , Vacina contra Coqueluche/imunologia , Receptores Toll-Like/imunologia , Vacinas/química , Vacinas Atenuadas/imunologiaRESUMO
Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Pneumocócicas/administração & dosagem , Vacinas Pneumocócicas/imunologia , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Células Cultivadas , Citocinas/metabolismo , Feminino , Vetores Genéticos , Lacticaseibacillus casei/genética , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neutrófilos/imunologia , Análise de Sequência de DNA , Baço/imunologia , Vacinas Sintéticas/genética , Fatores de Virulência/imunologiaRESUMO
Aluminum salts have been widely used in vaccine formulations and, after their introduction more than 80 years ago, only few vaccine formulations using new adjuvants were developed in the last two decades. Recent advances in the understanding of how innate mechanisms influence the adaptive immunity opened up the possibility for the development of new adjuvants in a more rational design. The purpose of this review is to discuss the recent advances in this field regarding the attempts to determine the molecular basis and the general mechanisms underlying the development of new adjuvants, with particular emphasis on the activation of receptors of innate immune recognition. One can anticipate that the use of these novel adjuvants will also provide a window of opportunities for the development of new vaccines.
Assuntos
Animais , Humanos , Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Vacinas/imunologia , Fatores de Virulência/imunologia , Adjuvantes Imunológicos/química , Compostos de Alumínio/imunologia , Imunidade Celular/imunologia , Vacina contra Coqueluche/imunologia , Receptores Toll-Like/imunologia , Vacinas Atenuadas/imunologia , Vacinas/químicaRESUMO
One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/administração & dosagem , Toxina da Cólera/administração & dosagem , Vacinas Pneumocócicas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Streptococcus pneumoniae/imunologia , Administração Intranasal , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Toxina da Cólera/metabolismo , Feminino , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/isolamento & purificação , Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Boca/microbiologia , Nasofaringe/microbiologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Proteínas de Choque Térmico/imunologia , Lipoproteínas/imunologia , Proteínas de Membrana Transportadoras , Vacinas Pneumocócicas/farmacologia , Streptococcus pneumoniae/imunologia , Adesinas Bacterianas , Animais , Anticorpos Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , DNA Bacteriano/genética , Proteínas de Choque Térmico/genética , Humanos , Imunidade Celular , Interferon gama/biossíntese , Interleucina-4/biossíntese , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Pneumocócicas/genética , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/genética , Células Th1/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologiaRESUMO
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.
Assuntos
Anticorpos Antibacterianos/biossíntese , Vacina BCG/imunologia , Proteínas de Bactérias/imunologia , Toxina Diftérica/imunologia , Vacinas Sintéticas/imunologia , Animais , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.