RESUMO
Consecutive lots of H5N1 (A/Vietnam/1194/2004 - NIBRG-14) split virion and whole virus vaccines were produced in a pilot-scale laboratory. The average yields of vaccine doses (15 microg HA) per egg were 0.57 doses for H5N1 split virion vaccine and 1.12 for H5N1 whole virus vaccine, compared to 2.09 doses for the seasonal H3N2 split virion vaccine. H5N1 split virion vaccine lots complied with WHO protein content criteria, while some lots of the H5N1 whole virus vaccine showed protein content per dose higher than the limit established. All lots of both vaccines showed ovalbumin (OVA) concentration below the recommended limit. Dose sparing strategies using adjuvant formulations using aluminum hydroxide (Al(OH)(3)) and monophosphoryl lipid A (MPLA) from Bordetella pertussis were tested in mice. Both 3.75 microg HA and 7.5 microg HA of H5N1 split virion vaccine with Al(OH)(3) or Al(OH)(3) plus MPLA in aqueous suspension showed higher hemagglutination-inhibition (HAI) titers when compared to the same vaccine dose without any adjuvant. Immunization with the H5N1 inactivated whole virus vaccine was also performed using 3.75 microg HA and HAI titers were higher than those induced by the split virion vaccine. Moreover, the use of Al(OH)(3) with MPLA as an emulsion induced a further increase in HAI titers.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Bordetella pertussis/química , Feminino , Testes de Inibição da Hemaglutinação , Vacinas contra Influenza/química , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Lipídeo A/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/análise , Vacinas de Produtos Inativados/química , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The world production capacity of influenza vaccines is a concern in face of the potential influenza pandemic. The use of adjuvants could increase several fold the current installed production capacity. Bordetella pertussis monophosphyl lipid A (MPLA) was produced by acid hydrolysis of LPS, obtained as a by-product of its removal from cellular pertussis vaccine, generating a product with 4 side chains. We have investigated different formulations including MPLA alone or combined with Al(OH)(3) as adjuvants for an inactivated split virion influenza vaccine. Our results demonstrate that MPLA at concentrations as low as 0.01 microg per dose of vaccine is effective, even with a 4-fold reduction of the regular vaccine dose, as measured by the induction of protective hemagglutination inhibition (HAI) titers. Al(OH)(3) can be combined with 0.01-10 microg MPLA, inducing even higher immune responses. Al(OH)(3) caused a drift of the immune response induced by the vaccine towards a Th2 profile, as evaluated by an increase in the IgG1:IgG2a ratio, while MPLA showed a more balanced response. Moreover, the use of MPLA and Al(OH)(3) combination led to the induction of the highest IgG levels together with the secretion of both IFN-gamma and IL-4. Although cell-mediated immune responses have not been usually taken into account for influenza vaccine formulations, they may be relevant for the induction of cross-protection as well as immunological memory for both inter-pandemic and pandemic influenza vaccines. Our results indicate that a more favorable profile of both humoral and cell-mediated immune responses may be obtained using the MPLA/Al(OH)(3) formulation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Bordetella pertussis/química , Vacinas contra Influenza/imunologia , Lipídeo A/análogos & derivados , Adjuvantes Imunológicos/isolamento & purificação , Hidróxido de Alumínio/farmacologia , Animais , Anticorpos Antivirais/sangue , Testes de Inibição da Hemaglutinação , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-4/metabolismo , Lipídeo A/isolamento & purificação , Lipídeo A/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
To add new insight to our previous work on the molecular epidemiology of Bordetella pertussis in Argentina, the prn and ptxS1 gene sequences and pulsed-field gel electrophoresis (PFGE) profiles of 57 clinical isolates obtained during two periods, 1969 to 1989 and 1997 to 2006, were analyzed. Non-vaccine-type ptxS1A was detected in isolates obtained since 1969. From 1989 on, a shift of predominance from the vaccine prn1 type to the nonvaccine prn2 type was observed. This was also reflected in a transition of PFGE group IV to group VI. These results show that nonvaccine B. pertussis strains are currently circulating. To analyze whether the observed genomic divergences between vaccine strains and clinical isolates have functional implications, protection assays using the intranasal mouse challenge model were performed. For such experiments, the clinical isolate B. pertussis 106 was selected as representative of circulating bacteria, since it came from the major group of the PFGE dendrogram (PFGE group VI). Groups of mice were immunized either with diphtheria-tetanus-whole-cell pertussis vaccine (ptxS1B prn1) or a vaccine prepared by us containing B. pertussis 106. Immunized mice were then challenged with a B. pertussis vaccine strain (Tohama, harboring ptxS1B and prn1) or the clinical isolate B. pertussis 106 (ptxS1A prn2). An adequate bacterial-elimination rate was observed only when mice were immunized and challenged with the same kind of strain. For further characterization, comparative proteomic profiling of enriched membrane proteins was done using three vaccine strains and the selected B. pertussis 106 clinical isolate. By matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, a total of 54 proteins were identified. This methodology allowed us to detect differing proteins among the four strains studied and, in particular, to distinguish the three vaccine strains from each other, as well as the vaccine strains from the clinical isolate. The differing proteins observed have cellular roles associated with amino acid and carbohydrate transport and metabolism. Some of them have been proposed as novel vaccine candidate proteins for other pathogens. Overall, the global strategy described here is presented as a good tool for the development of next-generation acellular vaccines.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/análise , Bordetella pertussis/química , Bordetella pertussis/genética , Toxina Pertussis/análise , Vacina contra Coqueluche , Fatores de Virulência de Bordetella/análise , Animais , Antígenos de Bactérias/imunologia , Argentina , Proteínas da Membrana Bacteriana Externa/genética , Bordetella pertussis/classificação , Bordetella pertussis/imunologia , Bordetella pertussis/isolamento & purificação , Contagem de Colônia Microbiana , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Esquemas de Imunização , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Toxina Pertussis/genética , Vacina contra Coqueluche/imunologia , Polimorfismo Genético , Proteômica , Fatores de Virulência de Bordetella/genética , Coqueluche/imunologia , Coqueluche/prevenção & controleRESUMO
Regulation of gene expression in response to local iron concentration is commonly observed in bacterial pathogens that face this nutrient limitation during host infection. In this study, a proteomic approach was used to analyze the differential protein expression of Bordetella pertussis under iron limitation. Whole cell lysates (WCL) and outer membrane fractions of bacteria grown either under iron-starvation or iron-excess conditions were analyzed by two-dimensional (2-D) gel electrophoresis. Statistical analysis revealed 36 proteins displaying differential expression, 9 with higher expression under iron-excess and 27 with increased expression under iron-starvation. These proteins were subjected to tryptic digestion and MALDI-TOF MS. Apart from those previously reported, we identified new low-iron-induced proteins that might help to explain the increased virulence of this phenotype. Additionally, we found evidence that at least one of the identified proteins, solely expressed under iron starvation, is highly immunogenic in infected individuals.
Assuntos
Proteínas de Bactérias/análise , Bordetella pertussis/química , Deficiências de Ferro , Proteoma/análise , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Bordetella pertussis/imunologia , Bordetella pertussis/metabolismo , Eletroforese em Gel Bidimensional , Análise Serial de Proteínas , Proteoma/imunologia , Proteômica , Tripsina/químicaRESUMO
This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-microm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg(+)) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 microm in diameter and 24 microm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.
Assuntos
Biofilmes/crescimento & desenvolvimento , Bordetella pertussis/citologia , Bordetella pertussis/fisiologia , Contagem de Colônia Microbiana/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Bordetella pertussis/química , Bordetella pertussis/isolamento & purificação , Proliferação de CélulasRESUMO
Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200-900 cm(-1) region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm(-1)) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.
Assuntos
Biofilmes/crescimento & desenvolvimento , Bordetella pertussis/química , Bordetella pertussis/crescimento & desenvolvimento , Lipopolissacarídeos/análise , Aderência Bacteriana , Técnicas Bacteriológicas , Bordetella pertussis/ultraestrutura , Meios de Cultura , Microesferas , Polipropilenos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Bordetella pertussis virulence-associated 30-, 32-, 90- and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30- and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bordetella pertussis/química , Bordetella pertussis/patogenicidade , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Dados de Sequência Molecular , Precursores de Proteínas/química , Alinhamento de Sequência , VirulênciaRESUMO
A simple procedure for elution in water of bacterial lipopolysaccharides (LPS) from sodium dodecyl sulfate-polyacrylamide gels is described. It consists of the combination of three principal steps: first, highly sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole (negative or reverse staining); second, washing of the individual LPS band in a solution of a zinc-complexing agent (e.g., 100 mM EDTA); and finally, elution of the LPS (100-200 microliters water for a 0.5-microgram LPS band) from gel microparticles for 3 h at room temperature. Using this procedure, we have successfully eluted a variety of LPS forms from Bordetella pertussis, Escherichia coli 0111:B4, E. coli K-235, Salmonella enteritidis, and Pseudomonas aeruginosa. Elution recovery of rough or semismooth LPS was about 70-80%, while that of smooth LPS was only about 10%. Eluted LPS was biologically active as tested by limulus amebocyte lysate and TNF-alpha assays.