RESUMO
OBJECTIVES: To develop and practically test high-precision femtosecond laser ablation models for dental hard tissue that are useful for detailed planning of automated laser dental restorative treatment. METHODS: Analytical models are proposed, derived, and demonstrated for practical calculation of ablation rates, ablation efficiency and ablated morphology of human dental enamel and dentin using femtosecond lasers. The models assume an effective optical attenuation coefficient for the irradiated material. To achieve ablation, it is necessary for the local energy density of the attenuated pulse in the hard tissue to surpass a predefined threshold that signifies the minimum energy density required for material ionization. A 1029 nm, 40 W carbide 275 fs laser was used to ablate sliced adult human teeth and generate the data necessary for testing the models. The volume of material removed, and the shape of the ablated channel were measured using optical profilometry. RESULTS: The models fit with the measured ablation efficiency curve against laser fluence for both enamel and dentin, correctly capturing the fluence for optimum ablation and the volume of ablated material per pulse. The detailed shapes of a 400-micrometer wide channel and a single-pulse width channel are accurately predicted using the superposition of the analytical result for a single pulse. CONCLUSIONS: The findings have value for planning automated dental restorative treatment using femtosecond lasers. The measurements and analysis give estimates of the optical properties of enamel and dentin irradiated with an infrared femtosecond laser at above-threshold fluence and the proposed models give insight into the physics of femtosecond laser processing of dental hard tissue.
Assuntos
Terapia a Laser , Dente , Humanos , Dentina/cirurgia , Lasers , LuzRESUMO
We investigated the effect of femtosecond (fs) laser ablation of enamel and dentin for different pulse wavelengths: infrared (1030 nm), green (515 nm), and ultra-violet (343 nm) and for different pulse separations to determine the optimal irradiation conditions for the precise removal of dental hard tissues with the absence of structural and compositional damage. The ablation rates and efficiencies were established for all three laser wavelengths for both enamel and dentin at room temperature without using any irrigation or cooling system, and the surfaces were assessed with optical and scanning electron microscopy, optical profilometry, and Raman spectroscopy. We demonstrated that 515 nm fs irradiation provides the highest rate and efficiency for ablation, followed by infrared. Finally, we explored the temperature variations inside the dental pulp during the laser procedures for all three wavelengths and showed that the maximum increase at the optimum conditions for both infrared and green irradiations was 5.5 °C, within the acceptable limit of temperature increase during conventional dental treatments. Ultra-violet irradiation significantly increased the internal temperature of the teeth, well above the acceptable limit, and caused severe damage to tooth structures. Thus, ultra-violet is not a compatible laser wavelength for femtosecond teeth ablation.
Assuntos
Dentina , Terapia a Laser , Dentina/efeitos da radiação , Lasers , Terapia a Laser/métodos , Temperatura , Esmalte DentárioRESUMO
High fluence focused femtosecond laser pulses were used to perform fast, high precision and minimally damaging cavity cutting of teeth at room temperature without using any irrigation or cooling system. The optimal ablation rates were established for both enamel and dentin, and the surfaces were assessed with optical and scanning electron microscopy, Raman spectroscopy and optical profilometry. No chemical change in the composition of enamel and dentin was observed. We explored temperature variations inside the dental pulp during the laser procedure and showed the maximum increase was 5.5°C, within the acceptable limit of temperature increase during conventional dental treatments.
RESUMO
Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development. IMPORTANCE The target pathogen of this study, Streptococcus pneumoniae, kills over 300,000 children <5 years of age every single year, and is the leading cause of pneumonia-associated mortality globally. While the capsular polysaccharide (CPS)-based vaccine Prevnar13 prevents serious illness caused by 13 serotypes, ongoing Prevnar13 use has driven the emergence of nonincluded serotypes as major causes of infection and disease. To overcome this issue, we have developed a next-generation pneumococcal vaccine conferring serotype-independent protection. This vaccine shows equivalent or superior efficacy to Prevnar13, and performance was heightened when our vaccine was administered with no adjuvant. These findings should be considered for similar vaccines in development, as the benefit of adjuvant is often assumed and its automatic inclusion may be limiting product efficacy, resulting in potential abandonment of viable vaccine candidates, or prolonging their time to clinic.
Assuntos
Anticorpos Antibacterianos , Infecções Pneumocócicas , Camundongos , Coelhos , Animais , Vacinas Pneumocócicas , Streptococcus pneumoniae , Vacinas Conjugadas , Sorogrupo , Infecções Pneumocócicas/prevenção & controleRESUMO
In recent years there has been increasing advocacy for highly immunogenic gamma-irradiated vaccines, several of which are currently in clinical or pre-clinical trials. Importantly, various methods of mathematical modelling and sterility testing are employed to ensure sterility. However, these methods are designed for materials with a low bioburden, such as food and pharmaceuticals. Consequently, current methods may not be reliable or applicable to estimate the irradiation dose required to sterilize microbiological preparations for vaccine purposes, where bioburden is deliberately high. In this study we investigated the applicability of current methods to calculate the sterilizing doses for different microbes. We generated inactivation curves that demonstrate single-hit and multiple-hit kinetics under different irradiation temperatures for high-titre preparations of pathogens with different genomic structures. Our data demonstrate that inactivation of viruses such as Influenza A virus, Zika virus, Semliki Forest virus and Newcastle Disease virus show single-hit kinetics following exposure to gamma-irradiation. In contrast, rotavirus inactivation shows multiple-hit kinetics and the sterilizing dose could not be calculated using current mathematical methods. Similarly, Streptococcus pneumoniae demonstrates multiple-hit kinetics. These variations in killing curves reveal an important gap in current mathematical formulae to determine sterility assurance levels. Here we propose a simple method to calculate the irradiation dose required for a single log10 reduction in bioburden (D10) value and sterilizing doses, incorporating both single- and multiple-hit kinetics, and taking into account the possible existence of a resistance shoulder for some pathogens following exposure to gamma-irradiation.
Assuntos
Raios gama , Modelos Teóricos , Doses de Radiação , Esterilização/métodos , Animais , Chlorocebus aethiops , Cães , Relação Dose-Resposta à Radiação , Cinética , Streptococcus pneumoniae , Temperatura , Células Vero , Zika virus/efeitos da radiação , Infecção por Zika virus/prevenção & controleRESUMO
The upper respiratory tract is continuously exposed to a vast array of potentially pathogenic viruses and bacteria. Influenza A virus (IAV) has particular synergism with the commensal bacterium Streptococcus pneumoniae in this niche, and co-infection exacerbates pathogenicity and causes significant mortality. However, it is not known whether this synergism is associated with a direct interaction between the two pathogens. We have previously reported that co-administration of a whole-inactivated IAV vaccine (γ-Flu) with a whole-inactivated pneumococcal vaccine (γ-PN) enhances pneumococcal-specific responses. In this study, we show that mucosal co-administration of γ-Flu and γ-PN similarly augments IAV-specific immunity, particularly tissue-resident memory cell responses in the lung. In addition, our in vitro analysis revealed that S. pneumoniae directly interacts with both γ-Flu and with live IAV, facilitating increased uptake by macrophages as well as increased infection of epithelial cells by IAV. These observations provide an additional explanation for the synergistic pathogenicity of IAV and S. pneumoniae, as well as heralding the prospect of exploiting the phenomenon to develop better vaccine strategies for both pathogens.
Assuntos
Imunidade , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Vacinas Pneumocócicas/imunologia , Animais , Coinfecção/imunologia , Coinfecção/prevenção & controle , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Células Epiteliais , Feminino , Humanos , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/administração & dosagem , Pulmão/imunologia , Macrófagos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/patogenicidade , Linfócitos T/imunologiaRESUMO
Existing capsular polysaccharide-based vaccines against pneumococcal disease are highly effective against vaccine-included serotypes, but they are unable to combat serotype replacement. We have developed a novel pneumococcal vaccine that confers serotype-independent protection, and could therefore constitute a "universal" vaccine formulation. This preparation is comprised of whole un-encapsulated pneumococci inactivated with gamma irradiation (γ-PN), and we have previously reported induction of cross-reactive immunity after nonadjuvanted intranasal vaccination. To further enhance vaccine immunogenicity and safety, we modified the pneumococcal vaccine strain to induce a stressed state during growth. Specifically, the substrate binding component of the psaBCA operon for manganese import was mutated to create a pneumococcal surface antigen A (psaA) defective vaccine strain. psaA mutation severely attenuated the growth of the vaccine strain in vitro without negatively affecting pneumococcal morphology, thereby enhancing vaccine safety. In addition, antibodies raised against vaccine preparations based on the modified strain [γ-PN(ΔPsaA)] showed more diversified reactivity to wild-type pneumococcal challenge strains compared to those induced by the original formulation. The modified vaccine also induced comparable protective TH 17 responses in the lung, and conferred greater protection against lethal heterologous pneumococcal challenge. Overall, the current study demonstrates successful refinement of a serotype-independent pneumococcal vaccine candidate to enhance safety and immunogenicity.
Assuntos
Adesinas Bacterianas/imunologia , Lipoproteínas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Adesinas Bacterianas/genética , Administração Intranasal , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Imunogenicidade da Vacina , Lipoproteínas/genética , Pulmão/citologia , Pulmão/imunologia , Camundongos , Mutação , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/administração & dosagem , Vacinas Pneumocócicas/efeitos adversos , Vacinas Pneumocócicas/genética , Streptococcus pneumoniae/genética , Células Th17/imunologia , Vacinação/métodos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologiaRESUMO
Rotavirus (RV) causes significant morbidity and mortality in developing countries, where children and infants are highly susceptible to severe disease symptoms. While live attenuated vaccines are available, reduced vaccine efficacy in developing countries illustrates the need for highly immunogenic alternative vaccines. Here, we studied the possible inactivation of RV using gamma(γ)-irradiation, and assessed the sterility and immunogenicity of γ-irradiated RV (γ-RV) as a novel vaccine candidate. Interestingly, the inactivation curve of RV did not show a log-linear regression following exposure to increased doses of γ-rays, and consequently the radiation dose required to achieve the internationally accepted Sterility Assurance Level could not be calculated. Nonetheless, we performed sterility testing based on serial passages of γ-RV, and our data clearly illustrate the lack of infectivity of γ-RV preparations irradiated with 50 kGy. In addition, we tested the immunogenicity of 50 kGy γ-RV in mice and our data illustrate the induction of strong RV-specific neutralising antibody responses following administration of γ-RV without using adjuvant. Therefore, whilst γ-RV may not constitute a replacement for current RV vaccines, this study represents a proof-of-concept that γ-irradiation can be applied to inactivate RV for vaccine purposes. Further investigation will be required to address whether γ-irradiation can be applied to improve safety and efficacy of existing live attenuated vaccines.
Assuntos
Raios gama , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus , Rotavirus/efeitos da radiação , Vacinas de Produtos Inativados , Inativação de Vírus/efeitos da radiação , Animais , Células Cultivadas , Chlorocebus aethiops , Feminino , Imunogenicidade da Vacina/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Rotavirus/imunologia , Vacinas contra Rotavirus/imunologia , Vacinas contra Rotavirus/uso terapêutico , Vacinas de Produtos Inativados/uso terapêutico , Células VeroRESUMO
Gamma-irradiation, particularly an irradiation dose of 50kGy, has been utilised widely to sterilise highly pathogenic agents such as Ebola, Marburg Virus, and Avian Influenza H5N1. We have reported previously that intranasal vaccination with a gamma-irradiated Influenza A virus vaccine (γ-Flu) results in cross-protective immunity. Considering the possible inclusion of highly pathogenic Influenza strains in future clinical development of γ-Flu, an irradiation dose of 50kGy may be used to enhance vaccine safety beyond the internationally accepted Sterility Assurance Level (SAL). Thus, we investigated the effect of irradiation conditions, including high irradiation doses, on the immunogenicity of γ-Flu. Our data confirm that irradiation at low temperatures (using dry-ice) is associated with reduced damage to viral structure compared with irradiation at room temperature. In addition, a single intranasal vaccination with γ-Flu irradiated on dry-ice with either 25 or 50kGy induced seroconversion and provided complete protection against lethal Influenza A challenge. Considering that low temperature is expected to reduce the protein damage associated with exposure to high irradiation doses, we titrated the vaccine dose to verify the efficacy of 50kGy γ-Flu. Our data demonstrate that exposure to 50kGy on dry-ice is associated with limited effect on vaccine immunogenicity, apparent only when using very low vaccine doses. Overall, our data highlight the immunogenicity of influenza virus irradiated at 50kGy for induction of high titre antibody and cytotoxic T-cell responses. This suggests these conditions are suitable for development of γ-Flu vaccines based on highly pathogenic Influenza A viruses.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza/efeitos da radiação , Infecções por Orthomyxoviridae/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Vacinação , Administração Intranasal , Animais , Cães , Relação Dose-Resposta Imunológica , Relação Dose-Resposta à Radiação , Feminino , Raios gama , Esquemas de Imunização , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza/administração & dosagem , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Análise de Sobrevida , Linfócitos T Citotóxicos/virologia , Potência de Vacina , Vacinas de Produtos InativadosRESUMO
Streptococcus pneumoniae and influenza are the world's foremost bacterial and viral respiratory pathogens. We have previously described a γ-irradiated influenza A virus (γ-FLU) vaccine that provides cross-protective immunity against heterosubtypic infections. More recently, we reported a novel non-adjuvanted γ-irradiated S pneumoniae (γ-PN) vaccine that elicits serotype-independent protection. Considering the clinical synergism of both pathogens, combination of a serotype-independent pneumococcal vaccine with a broad-spectrum influenza vaccine to protect against both infections would have a considerable clinical impact. In the present study, we co-immunized C57BL/6 mice intranasally (IN) with a mixture of γ-PN (whole inactivated cells) and γ-FLU (whole inactivated virions) and examined protective efficacy. Co-immunization enhanced γ-PN vaccine efficacy against virulent pneumococcal challenge, which was dependent on CD4+ T-cell responses. In contrast, vaccination with γ-PN alone, co-immunization enhanced pneumococcal-specific effector T-helper 17 cell (Th17) and Th1 memory cell, promoted development of CD4+ tissue-resident memory (TRM) cells and enhanced Pneumococcus-specific antibody responses. Furthermore, co-immunization elicited significant protection against lethal influenza challenge, as well as against co-infection with both influenza and S pneumoniae. This is the first report showing the synergistic effect of combining whole cell and whole virion vaccines to both S pneumoniae and influenza as a single vaccine to protect against individual and co-infection, without compromising pathogen-specific immunity.
Assuntos
Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Administração Intranasal , Animais , Formação de Anticorpos , Humanos , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/imunologia , VacinaçãoRESUMO
Generating a pneumococcal vaccine that is serotype independent and cost effective remains a global challenge. γ-Irradiation has been used widely to sterilize biological products. It can also be utilized as an inactivation technique to generate whole-cell bacterial and viral vaccines with minimal impact on pathogen structure and antigenic determinants. In the present study, we utilized γ-irradiation to inactivate an un-encapsulated Streptococcus pneumoniae strain Rx1 with an unmarked deletion of the autolysin gene lytA and with the pneumolysin gene ply replaced with an allele encoding a non-toxic pneumolysoid (PdT) (designated γ-PN vaccine). Intranasal vaccination of C57BL/6 mice with γ-PN was shown to elicit serotype-independent protection in lethal challenge models of pneumococcal pneumonia and sepsis. Vaccine efficacy was shown to be reliant on B-cells and interleukin (IL)-17A responses. Interestingly, immunization promoted IL-17 production by innate cells not T helper 17 (Th17) cells. These data are the first to report the development of a non-adjuvanted intranasal γ-irradiated pneumococcal vaccine that generates effective serotype-independent protection, which is mediated by both humoral and innate IL-17 responses.
Assuntos
Linfócitos B/imunologia , Raios gama , Imunidade Inata , Interleucina-17/metabolismo , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/efeitos da radiação , Vacinação , Administração Intranasal , Animais , Linfócitos T CD4-Positivos/imunologia , Imunidade Inata/imunologia , Memória Imunológica , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/complicações , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Sepse/complicações , Sepse/imunologia , Sepse/microbiologia , Sepse/prevenção & controle , Sorotipagem , Streptococcus pneumoniae/classificação , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
Escherichia coli's heat-labile enterotoxin (Etx) and its non-toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co-administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC-II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells.
Assuntos
Apresentação de Antígeno/imunologia , Toxinas Bacterianas/farmacologia , Células Dendríticas/imunologia , Enterotoxinas/farmacologia , Proteínas de Escherichia coli/farmacologia , Subunidades Proteicas/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Células da Medula Óssea/citologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Feminino , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Baço/citologiaRESUMO
Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.
Assuntos
Toxinas Bacterianas/metabolismo , Toxina da Cólera/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Intestino Delgado/enzimologia , Peptídeo Hidrolases/metabolismo , Lectinas de Plantas/metabolismo , Ulex/metabolismo , Vibrio cholerae/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxina da Cólera/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Feminino , Conteúdo Gastrointestinal/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Lectinas de Plantas/genética , Inibidores de Proteases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Epitopos/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/imunologia , Região Variável de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Especificidade de Anticorpos/genética , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Reações Cruzadas/genética , Reações Cruzadas/imunologia , Enterotoxinas/análise , Enterotoxinas/genética , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/química , Epitopos/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/genética , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/genética , Gangliosídeo G(M1)/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Estrutura Quaternária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de DNA/métodosRESUMO
A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations.
Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli/química , Gangliosídeo G(M1)/síntese química , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Motivos de Aminoácidos , Sítios de Ligação , Escherichia coli/química , Escherichia coli/metabolismo , Fluorescência , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Ligantes , Fragmentos de Peptídeos/químicaRESUMO
Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.
Assuntos
Enterotoxinas/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Animais , Anticorpos Monoclonais/química , Toxinas Bacterianas/química , Sítios de Ligação , Relação Dose-Resposta a Droga , Enterotoxinas/química , Proteínas de Escherichia coli/química , Hibridomas/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule that acts both as an adjuvant and to stimulate immune deviation processes, resulting in the suppression of Th1-associated inflammatory responses. The ability of EtxB to alter immune reactivity is dependent on its ability to modulate immune cell function through binding to cell surface molecules, the principal receptor of which is the ubiquitous GM1-ganglioside. EtxB activates B cells and antigen-presenting cells and induces the selective apoptosis of murine CD8+ T cells. We postulated that these effects are mediated by the induction of intracellular signaling pathways following EtxB-receptor interaction. We have previously shown that CD8+ T-cell apoptosis induced by EtxB results from the activation of the transcription factor NF-kappaB and caspases. Here we report that while caspase activity is required for apoptosis, additional features of cell death are caspase independent. EtxB induces a rapid loss of mitochondrial membrane potential and cell viability that are unaffected by caspase inhibitors. In addition, our data suggest that these processes are independent of the activity of Bax and Bcl-2 but are mediated by nitric oxide synthase.
Assuntos
Apoptose/efeitos dos fármacos , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Caspases/metabolismo , Enterotoxinas/química , Enterotoxinas/farmacologia , Proteínas de Escherichia coli , Subunidades Proteicas/química , Subunidades Proteicas/farmacologia , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Inibidores de Caspase , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Escherichia coli , Feminino , Guanidinas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Força Próton-Motriz/efeitos dos fármacos , Proteína X Associada a bcl-2RESUMO
The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.
Assuntos
Enterotoxinas/química , Complexo Principal de Histocompatibilidade , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Antibacterianos/farmacologia , Apresentação de Antígeno , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão , Citosol/metabolismo , Células Dendríticas/citologia , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Epitopos/química , Escherichia coli/metabolismo , Filipina/farmacologia , Complexo de Golgi/metabolismo , Caranguejos Ferradura/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fagocitose , Inibidores de Proteases/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Tempo , Vibrio/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Cholera toxin (Ctx) from Vibrio cholerae and its closely related homologue, heat-labile enterotoxin (Etx) from Escherichia coli have become superb tools for illuminating pathways of cellular trafficking and immune cell function. These bacterial protein toxins should be viewed as conglomerates of highly evolved, multi-functional elements equipped to engage the trafficking and signalling machineries of cells. Ctx and Etx are members of a larger family of A-B toxins of bacterial (and plant) origin that are comprised of structurally and functionally distinct enzymatically active A and receptor-binding B sub-units or domains. Intoxication of mammalian cells by Ctx and Etx involves B pentamer-mediated receptor binding and entry into a vesicular pathway, followed by translocation of the enzymatic A1 domain of the A sub-unit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase and a sequence of events culminating in life-threatening diarrhoeal disease. Importantly, Ctx and Etx also have the capacity to induce a wide spectrum of remarkable immunological processes. With respect to the latter, it has been found that these toxins activate signalling pathways that modulate the immune system. This review explores the complexities of the cellular interactions that are engaged by these bacterial protein toxins, and highlights some of the new insights to have recently emerged.
Assuntos
Proteínas de Bactérias , Toxina da Cólera , Proteínas de Escherichia coli , Escherichia coli/química , Vibrio cholerae/química , Adenilil Ciclases/metabolismo , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Transporte Biológico Ativo/fisiologia , Toxina da Cólera/química , Toxina da Cólera/imunologia , Toxina da Cólera/metabolismo , Diarreia/fisiopatologia , Enterotoxinas/química , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Humanos , Sistema Imunitário/fisiopatologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (alpha2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.