RESUMEN
In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 µg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/biosíntesis , Gripe Humana/prevención & control , Pandemias/prevención & control , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/inmunología , Composición de Medicamentos/métodos , Composición de Medicamentos/estadística & datos numéricos , Industria Farmacéutica/normas , Femenino , Humanos , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Gripe Humana/inmunología , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Vacunas de Productos Inactivados/biosíntesis , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificaciónRESUMEN
A number of adjuvant formulations were assayed in mice immunized with 3.75 µg of A/California/7/2009 (H1N1) pdm09 influenza vaccine with vitamins A, D and/or E in emulsions or B2 and/or B9 combined with Bordetella pertussis MPLA and/or alum as adjuvants. Squalene was used as positive control, as well as MPLA with alum. The immune response was evaluated by a panel of tests, including a hemagglutination inhibition (HAI) test, ELISA for IgG, IgG1, and IgG2a and IFN-γ, IL-2, IL-6 and IL-10 quantification in splenocyte culture supernatant after stimulus with influenza antigen. Immunological memory was evaluated using a 1/10 dose booster 60 days after the first immunization followed by assessment of the response by HAI, IgG ELISA, and determination of the antibody affinity index. The highest increases in HAI, IgG1 and IgG2a titers were obtained with the adjuvant combinations containing vitamin E, or the hydrophilic combinations containing MPLA and alum or B2 and alum. The IgG1/IgG2a ratio indicates that the response to the combination of B2 with alum would have more Th2 character than the combination of MPLA with alum. In an assay to investigate the memory response, a significant increase in HAI titer was observed with a booster vaccine dose at 60 days after immunization with vaccines containing MPLA with alum or B2 with alum. Overall, of the 27 adjuvant combinations, MPLA with alum and B2 with alum were the most promising adjuvants to be evaluated in humans.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra la Influenza/inmunología , Vitaminas/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Antígenos Bacterianos/administración & dosificación , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Pruebas de Inhibición de Hemaglutinación , Memoria Inmunológica , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Leucocitos Mononucleares/inmunología , Masculino , Ratones Endogámicos BALB C , Escualeno/administración & dosificaciónRESUMEN
Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.
Asunto(s)
Cromatografía de Afinidad/métodos , Cobre/química , Factor VIII/aislamiento & purificación , Factor VIII/química , Factor VIII/metabolismo , Humanos , Modelos MolecularesRESUMEN
In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Proteínas de la Membrana/inmunología , Pichia/inmunología , Plasmodium vivax/inmunología , Proteínas Protozoarias/inmunología , Levaduras/inmunología , Adyuvantes Inmunológicos/genética , Animales , Formación de Anticuerpos/inmunología , Antígenos de Protozoos/genética , Femenino , Humanos , Inmunización/métodos , Inmunoglobulina G/inmunología , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Malaria Vivax/genética , Malaria Vivax/inmunología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Pichia/genética , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Levaduras/genéticaRESUMEN
Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr(-)) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.
Asunto(s)
Células CHO/citología , Células CHO/fisiología , Glucosilceramidasa/biosíntesis , Glucosilceramidasa/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Animales , Clonación Molecular , Cricetinae , Cricetulus , Glucosilceramidasa/aislamiento & purificación , HumanosRESUMEN
Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.
Asunto(s)
Biotecnología/métodos , Factor VIII/aislamiento & purificación , Plasma/química , Tecnología Farmacéutica/métodos , Resinas de Intercambio Aniónico , Cromatografía en Gel , Cromatografía por Intercambio Iónico/métodos , HumanosRESUMEN
BACKGROUND: The Butantan Institute has manufactured a lyophilised tetravalent live-attenuated dengue vaccine Butantan-DV, which is analogous to the US National Institutes of Health (NIH) TV003 admixture. We aimed to assess the safety and immunogenicity of Butantan-DV. METHODS: We did a two-step, double-blind, randomised placebo-controlled phase 2 trial at two clinical sites in São Paulo, Brazil. We recruited healthy volunteers aged 18-59 years; pregnant women, individuals with a history of neurological, heart, lung, liver or kidney disease, diabetes, cancer, or autoimmune diseases, and individuals with HIV or hepatitis C were excluded. Step A was designed as a small bridge-study between Butantan-DV and TV003 in DENV-naive participants. In step A, we planned to randomly assign 50 dengue virus (DENV)-naive individuals to receive two doses of Butantan-DV, TV003, or placebo, given 6 months apart. In step B, we planned to randomly assign 250 participants (DENV-naive and DENV-exposed) to receive one dose of Butantan-DV or placebo. Participants were randomly assigned, by computer-generated block randomisation (block sizes of five); participants in step A were randomly assigned (2:2:1) to receive Butantan-DV, TV003, or placebo and participants in step B were randomly assigned (4:1) to receive Butantan-DV or placebo. Participants and study staff were unaware of treatment allocation. The primary safety outcome was the frequency of solicited and unsolicited local and systemic adverse reactions within 21 days of the first vaccination, analysed by intention to treat. The primary immunogenicity outcome was seroconversion rates of the DENV-1-4 serotypes measured 91 days after the first vaccination, analysed in the per-protocol population, which included all participants in step A, and all participants included in step B who completed all study visits with serology sample collection. This trial is registered with ClinicalTrials.gov, NCT01696422. FINDINGS: Between Nov 5, 2013, and Sept 21, 2015, 300 individuals were enrolled and randomly assigned: 155 (52%) DENV-naive participants and 145 (48%) DENV-exposed participants. Of the 155 DENV-naive participants, 97 (63%) received Butantan-DV, 17 (11%) received TV003, and 41 (27%) received placebo. Of the 145 DENV-exposed participants, 113 (78%) received Butantan-DV, three (2%) received TV003, and 29 (20%) received placebo. Butantan-DV and TV003 were both immunogenic, well-tolerated, and no serious adverse reactions were observed. In step A, rash was the most frequent adverse event (16 [845] of 19 participants in the Butantan-DV group and 13 [76%] of 17 participants in the TV003 group). Viraemia was similar between the Butantan-DV and TV003 groups. Of the 85 DENV-naive participants in the Butantan-DV group who attended all visits for sample collection for seroconversion analysis and thus were included in the per-protocol analysis population, 74 (87%) achieved seroconversion to DENV-1, 78 (92%) to DENV-2, 65 (76%) to DENV-3, and 76 (89%) to DENV-4. Of the 101 DENV-exposed participants in the Butantan-DV group who attended all visits for sample collection for seroconversion analysis, 82 (81%) achieved seroconversion to DENV-1, 79 (78%) to DENV-2, 83 (82%) to DENV-3, and 78 (77%) to DENV-4. INTERPRETATION: Butantan-DV and TV003 were safe and induced robust, balanced neutralising antibody responses against the four DENV serotypes. Efficacy evaluation of the Butantan-DV vaccine is ongoing. FUNDING: Intramural Research Program US NIH National Institute of Allergy and Infectious Diseases, Brazilian National Bank for Economic and Social Development, Fundação de Amparo à Pesquisa do Estado de São Paulo, and Fundação Butantan.
Asunto(s)
Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Inmunogenicidad Vacunal , Vacunas Atenuadas/inmunología , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Brasil , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Seroconversión , Vacunación , Adulto JovenRESUMEN
Polymeric microspheres containing diphtheria and tetanus toxoids were prepared without protein stabilizers. A vaccine containing 2 Lf(tetanus) and 0.4 Lf(diphtheria) was injected either in BALB/c mice or in guinea-pigs. As control, a group received the alum-adsorbed unencapsulated toxoids. In mice, on day 44 one group and control received a booster and at day 111 the other group received the same booster dose. Before de booster, all groups had very low neutralizing antibodies, as determined by Toxin binding inhibition assay. One week after booster all groups had high antibody titers, especially those immunized with microencapsulated vaccine, which were at least 5 times higher than those immunized with alum vaccine for both antigens. Besides, guinea pigs receiving lower dose had antibodies titers as high as 60 UI/mL, and 30 times higher than those immunized with alum vaccine. Therefore by using an encapsulated vaccine without any kind of protein stabilizer we were able to induce in vivo protective responses irrespective of observed in vitro protein degradation by HPLC. Manipulating the vaccination schedule at the same time to the toxoids encapsulation does not only increase the antibody titers but also their specificity.
Asunto(s)
Vacuna contra Difteria y Tétanos/administración & dosificación , Vacuna contra Difteria y Tétanos/inmunología , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Adyuvantes Inmunológicos , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Disponibilidad Biológica , Toxoide Diftérico/administración & dosificación , Toxoide Diftérico/inmunología , Toxoide Diftérico/farmacocinética , Vacuna contra Difteria y Tétanos/química , Composición de Medicamentos , Femenino , Cobayas , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Toxoide Tetánico/administración & dosificación , Toxoide Tetánico/inmunología , Toxoide Tetánico/farmacocinética , Vacunación/métodosRESUMEN
The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-gamma production and an increase in both IFN-gamma(+) and TNF-alpha(+)-CD4(+)-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.
Asunto(s)
Bordetella pertussis/inmunología , Mycobacterium bovis/genética , Toxina del Pertussis/inmunología , Vacuna contra la Tos Ferina/inmunología , Tos Ferina/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Bordetella pertussis/genética , Linfocitos T CD4-Positivos/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Humanos , Recién Nacido , Interferón gamma/biosíntesis , Ratones , Toxina del Pertussis/genética , Vacuna contra la Tos Ferina/genética , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/biosíntesis , Tos Ferina/inmunologíaRESUMEN
Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of approximately 7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 microg porcine aprotinin inhibited 6 microg trypsin whereas 1 microg commercial soybean inhibitor inhibited only 1 microg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.
Asunto(s)
Aprotinina/genética , Aprotinina/aislamiento & purificación , Pulmón/metabolismo , Secuencia de Aminoácidos , Animales , Aprotinina/metabolismo , Secuencia de Bases , Bovinos , Cromatografía de Afinidad , ADN/genética , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Porcinos , Tripsina/metabolismoRESUMEN
While the immunogenic potential of the vaccination against infectious diseases was extensively shown, data on the safety assessment of recombinant proteins in vaccine formulations administered during pregnancy are still scarce. In the current study, the antigenicity of a vaccine against leishmaniasis (based on Leishmania braziliensis recombinant protein peroxidoxin) during pregnancy and possible maternal reproductive outcomes and fetal anomalies after immunization with a leishmanial vaccine or adjuvant alone (Bordetella pertussis derived MPLA adjuvant) were assessed. Rats were mated and allocated in three groups: Control-rats received saline; Adjuvant-rats received the adjuvant MPLA, and Vaccine-rats received the combination of MPLA and peroxidoxin. The administration was subcutaneously at the dorsal region, three times (days 0, 7, 14 of pregnancy). On day 21 of pregnancy, all rats were bled for biochemical and immunological measurements. The gravid uterus was weighed with its contents, and the fetuses were analyzed. The immunization with peroxidoxin induced a significant production of circulating IgG levels compared to other groups but caused a significant in post-implantation loss (14.7%) when compared to Control (5.0%) and Adjuvant (4.4%) groups. Furthermore, a significantly high rate of fetal visceral anomalies, such as hydronephrosis and convoluted ureter, was also observed in animals that received vaccine when compared to Control or Adjuvant groups. These data indicate the importance of safety evaluation of vaccines during pregnancy and the limited use of peroxidoxin administration during pregnancy. More importantly, the safety monitoring of immunization with MPLA derived from Bordetella pertussis demonstrated no reproductive outcomes associated with adjuvant administration, suggesting its safe use during pregnancy.
Asunto(s)
Pérdida del Embrión/inducido químicamente , Feto/anomalías , Leishmania braziliensis , Vacunas contra la Leishmaniasis/efectos adversos , Exposición Materna/efectos adversos , Modelos Biológicos , Peroxirredoxinas/efectos adversos , Proteínas Protozoarias/efectos adversos , Animales , Anticuerpos Antiprotozoarios/inmunología , Evaluación Preclínica de Medicamentos , Femenino , Feto/inmunología , Inmunoglobulina G/inmunología , Vacunas contra la Leishmaniasis/inmunología , Vacunas contra la Leishmaniasis/farmacología , Peroxirredoxinas/inmunología , Peroxirredoxinas/farmacología , Embarazo , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/farmacología , RatasRESUMEN
PURPOSE: To study the immunogenicity and the stability of the porcine pulmonary surfactant preparation produced by the Instituto Butantan. METHOD: Immunogenicity assay: Sixteen New-Zealand-White rabbits (1000 g body weight) were divided into 4 study groups. Each group was assigned to receive either a) Butantan surfactant, b) Survanta (Abbott Laboratories), c) Curosurf (Farmalab Chiesi), or d) no surfactant. The surfactants were administered intratracheally, and the animals were collected immediately before and 60 and 180 days after surfactant administration. Sera were assayed for the presence of antisurfactant antibodies by enzyme-linked immunosorbent assay (ELISA). Stability assay: The Butantan surfactant used in this assay had been stored for one year in the refrigerator (4 to 8 degrees C) and its stability was evaluated in distinct assay conditions using a premature rabbit model. RESULTS: Immunogenicity assay: None of the surfactants analyzed triggered antibody immune responses against their components in any of the animals. Stability assay: The results of this study demonstrate that Butantan surfactant was as effective as Curosurf when both were submitted to the adverse circumstance of short- and long-term storage at room temperature. A similar level of efficacy for the Butantan surfactant, as compared to Curosurf was demonstrated by the pulmonary dynamic compliance, ventilatory pressure, and pressure-volume curve results. CONCLUSION: The results of our study demonstrate that Butantan surfactant may be a suitable alternative for surfactant replacement therapy.
Asunto(s)
Surfactantes Pulmonares/química , Surfactantes Pulmonares/inmunología , Animales , Animales Recién Nacidos , Femenino , Modelos Animales , Embarazo , Proteínas Asociadas a Surfactante Pulmonar/inmunología , Surfactantes Pulmonares/uso terapéutico , Conejos , Enfermedades Respiratorias/tratamiento farmacológico , Porcinos , Factores de TiempoRESUMEN
In areas were human visceral leishmaniasis (VL) is endemic, the domestic dog is the main parasite reservoir in the infectious cycle of Leishmania infantum. Development of prophylactic strategies to lower the parasite burden in dogs would reduce sand fly transmission thus lowering the incidence of zoonotic VL. Here we demonstrate that vaccination of dogs with a recombinant 14kDa polypeptide of L. infantum nuclear transport factor 2 (Li-ntf2) mixed with adjuvant BpMPLA-SE resulted in the production of specific anti-Li-ntf2 IgG antibodies as well as IFN-γ release by the animals' peripheral blood mononuclear cells stimulated with the antigen. In addition, immunization with this single and small 14kDa poplypeptide resulted in protracted progression of the infection of the animals after challenging with a high dose of virulent L. infantum. Five months after challenge the parasite load was lower in the bone marrow of immunized dogs compared to non-immunized animals. The antibody response to K39, a marker of active VL, at ten months after challenge was strong and significantly higher in the control dogs than in vaccinated animals. At the study termination vaccinated animals showed significantly more liver granulomas and lymphoid hyperplasia than non-vaccinated animals, which are both histological markers of resistance to infection. Together, these results indicate that the 14kDa polypeptide is an attractive protective molecule that can be easily incorporated in a leishmanial polyprotein vaccine candidate to augment/complement the overall protective efficacy of the final product.
RESUMEN
A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60 % of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Neisseria meningitidis/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Brasil , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C3H , Peso Molecular , Neisseria meningitidis Serogrupo B/inmunología , Neisseria meningitidis Serogrupo C/inmunología , Serotipificación , VacunaciónRESUMEN
Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached approximately 1.8 g of DCW/L, while polysaccharide concentration was about approximately 132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.
Asunto(s)
Infecciones por Haemophilus/metabolismo , Vacunas contra Haemophilus/biosíntesis , Haemophilus influenzae tipo b/metabolismo , Polisacáridos Bacterianos/biosíntesis , Cápsulas Bacterianas , Técnicas Bacteriológicas , Biomasa , Reactores Biológicos , División Celular , Fermentación , Glucosa/metabolismo , Haemophilus influenzae tipo b/crecimiento & desarrollo , Oxígeno/metabolismoRESUMEN
Pneumococcal surface protein A (PspA) is a candidate antigen for the composition of protein-based vaccines against Streptococcus pneumoniae. While searching for efficient adjuvants for PspA-based vaccines, our group has described the potential of combining PspA with the whole-cell pertussis vaccine (wP). When given to mice through the nasal route, a formulation composed of PspA from clade 5 (PspA5) and wP (PspA5-wP) induced high levels of antibodies and protection against challenges with different pneumococcal strains. PspA5-wP also induced the secretion of interleukin 17 (IL-17) by splenocytes and the infiltration of leukocytes in the lungs after challenge. Here, we show that protection against a pneumococcal invasive challenge was completely abrogated in µMT(-/-) mice, which are deficient in the maturation of B cells, illustrating the importance of antibodies in the survival elicited by the PspA5-wP vaccine. Moreover, passive immunization showed that IgG purified from the sera of mice immunized with PspA5-wP conferred significant protection to naive mice, whereas the respective F(ab')2 did not. Additionally, in vivo depletion of complement abolished protection against the pneumococcal challenge. The combination of PspA5 with wild-type or mutant Bordetella pertussis strains or with purified components showed that the pertussis toxin (PT)-containing formulations induced the highest levels of antibodies and protection. This suggests that the adjuvant activity of wP in the PspA5 model is mediated at least in part by PT. The sera from mice immunized with such formulations displayed high IgG binding and induction of complement deposition on the pneumococcal surface in vitro, which is consistent with the in vivo results.
Asunto(s)
Proteínas Bacterianas/inmunología , Toxina del Pertussis/inmunología , Infecciones Neumocócicas/inmunología , Vacunas Neumococicas/inmunología , Streptococcus pneumoniae/inmunología , Adyuvantes Inmunológicos , Administración Intranasal , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Bordetella pertussis/inmunología , Proteínas del Sistema Complemento/inmunología , Inmunización Pasiva , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/inmunología , Interleucina-17/metabolismo , Leucocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Vacuna contra la Tos Ferina/inmunología , Infecciones Neumocócicas/prevención & control , Receptores Inmunológicos/inmunología , Proteínas Recombinantes/inmunología , Vacunas Estreptocócicas/inmunología , VacunaciónRESUMEN
The Pneumococcal Surface Protein A (PspA) is a promising candidate for the composition of a protein vaccine against Streptococcus pneumoniae. We have previously shown that the whole cell Bordetella pertussis vaccine (wP) is a good adjuvant to PspA, inducing protective responses against pneumococcal infection in mice. In Brazil, wP is administered to children, formulated with diphtheria and tetanus toxoids (DTPw) and aluminum hydroxide (alum) as adjuvant. A single subcutaneous dose of PspA5-DTPlow (a formulation containing PspA from clade 5 and a new generation DTPw, containing low levels of B. pertussis LPS and Alum) induced high levels of systemic anti-PspA5 antibodies in mice and conferred protection against respiratory lethal challenges with two different pneumococcal strains. Here we evaluate the mucosal immune responses against PspA5 as well as the immune responses against the DTP antigens in mice vaccinated with PspA5-DTPlow. Subcutaneous immunization of mice with PspA5-DTPlow induced high levels of anti-PspA5 IgG in the airways but no IgA. In addition, no differences in the influx of cells to the respiratory mucosa, after the challenge, were observed in vaccinated mice, when compared with control mice. The levels of circulating anti-pertussis, -tetanus and -diphtheria antibodies were equivalent in mice vaccinated with DTPlow or PspA5-DTPlow. Antibodies induced by DTPlow or PspA5-DTPlow showed similar ability to neutralize the cytotoxic effects of the diphtheria toxin on Vero cells. Furthermore, combination with PspA5 did not affect protection against B. pertussis and tetanus toxin challenges in mice. Our results support the proposal for a combined PspA-DTP vaccine.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Inmunidad Mucosa/inmunología , Lipopolisacáridos/inmunología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Compuestos de Alumbre , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Bordetella pertussis/inmunología , Chlorocebus aethiops , Toxina Diftérica/antagonistas & inhibidores , Toxina Diftérica/inmunología , Femenino , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Toxina del Pertussis/antagonistas & inhibidores , Toxina del Pertussis/inmunología , Infecciones Neumocócicas/inmunología , Vacunas Neumococicas/administración & dosificación , Mucosa Respiratoria/inmunología , Streptococcus pneumoniae/inmunología , Toxina Tetánica/antagonistas & inhibidores , Toxina Tetánica/inmunología , Vacunación , Células VeroRESUMEN
We conducted a phase I, double-blind, placebo-controlled trial to evaluate a new 5-valent oral rotavirus vaccine's safety and immunogenicity profiles. Subjects were randomly assigned to receive 3 orally administered doses of a live-attenuated human-bovine (UK) reassortant rotavirus vaccine, containing five viral antigens (G1, G2, G3, G4 and G9), or a placebo. The frequency and severity of adverse events were assessed. Immunogenicity was evaluated by the titers of anti-rotavirus IgA and the presence of neutralizing antibodies anti-rotavirus. No severe adverse events were observed. There was no difference in the frequency of mild adverse events between experimental and control groups. The proportion of seroconversion was consistently higher in the vaccine group, for all serotypes, after each one of the doses. The 5-valent vaccine has shown a good profile of safety and immunogenicity in this small sample of adult volunteers.
Asunto(s)
Vacunas contra Rotavirus/efectos adversos , Vacunas contra Rotavirus/inmunología , Administración Oral , Adolescente , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Método Doble Ciego , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Humanos , Inmunoglobulina A/sangre , Masculino , Placebos/administración & dosificación , Vacunas contra Rotavirus/administración & dosificación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Adulto JovenRESUMEN
An improved whole cell pertussis vaccine, designated as Plow, which is low in endotoxicity due to a chemical extraction of lipo-oligosaccharide (LOS) from the outer membrane, was evaluated for safety, immunogenicity and potency, comparatively to a traditional whole cell pertussis vaccine. Current whole cell pertussis vaccines are effective but contain large quantities of endotoxin and consequently display local and systemic adverse reactions after administration. Endotoxin is highly inflammatory and contributes considerably to the reactogenicity as well as the potency of these vaccines. In contrast, acellular pertussis vaccines hardly contain endotoxin and are significantly less reactogenic, but their elevated costs limit their global use, especially in developing countries. In this paper, bulk products of Plow and a traditional whole cell vaccine, formulated as plain monocomponents or combined with diphtheria and tetanus toxoids (DTPlow or DTP, respectively) were compared by in vitro and in vivo assays. Chemical extraction of LOS resulted in a significant decrease in endotoxin content (20%) and a striking decline in endotoxin related toxicity (up to 97%), depending on the used in vitro or in vivo test. The LOS extraction did not affect the integrity of the product and, more importantly, did not affect the potency and/or stability of DTPlow. Moreover, hardly any differences in antibody and T-cell responses were observed. The development of Plow is a significant improvement regarding the endotoxicity of whole cell pertussis vaccines and therefore a promising and affordable alternative to currently available whole cell or acellular pertussis vaccines for developing countries.
Asunto(s)
Endotoxinas/aislamiento & purificación , Vacuna contra la Tos Ferina/efectos adversos , Vacuna contra la Tos Ferina/inmunología , Potencia de la Vacuna , Animales , Estabilidad de Medicamentos , Endotoxinas/análisis , Femenino , Ratones , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/química , ConejosRESUMEN
Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.