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1.
Infect Immun ; 88(4)2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-31932330

RESUMO

The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro-grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro-grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.

2.
Front Immunol ; 10: 2214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31616417

RESUMO

Immaturity of the immune system contributes to poor vaccine responses in early life. Germinal center (GC) activation is limited due to poorly developed follicular dendritic cells (FDC), causing generation of few antibody-secreting cells (ASCs) with limited survival and transient antibody responses. Herein, we compared the potential of five adjuvants, namely LT-K63, mmCT, MF59, IC31, and alum to overcome limitations of the neonatal immune system and to enhance and prolong responses of neonatal mice to a pneumococcal conjugate vaccine Pnc1-TT. The adjuvants LT-K63, mmCT, MF59, and IC31 significantly enhanced GC formation and FDC maturation in neonatal mice when co-administered with Pnc1-TT. This enhanced GC induction correlated with significantly enhanced vaccine-specific ASCs by LT-K63, mmCT, and MF59 in spleen 14 days after immunization. Furthermore, mmCT, MF59, and IC31 prolonged the induction of vaccine-specific ASCs in spleen and increased their persistence in bone marrow up to 9 weeks after immunization, as previously shown for LT-K63. Accordingly, serum Abs persisted above protective levels against pneumococcal bacteremia and pneumonia. In contrast, alum only enhanced the primary induction of vaccine-specific IgG Abs, which was transient. Our comparative study demonstrated that, in contrast to alum, LT-K63, mmCT, MF59, and IC31 can overcome limitations of the neonatal immune system and enhance both induction and persistence of protective immune response when administered with Pnc1-TT. These adjuvants are promising candidates for early life vaccination.

3.
Front Immunol ; 9: 381, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29541075

RESUMO

Neonates and infants are more vulnerable to infections and show reduced responses to vaccination. Consequently, repeated immunizations are required to induce protection and early life vaccines against major pathogens such as influenza are yet unavailable. Formulating antigens with potent adjuvants, including immunostimulators and delivery systems, is a demonstrated approach to enhance vaccine efficacy. Yet, adjuvants effective in adults may not meet the specific requirements for activating the early life immune system. Here, we assessed the neonatal adjuvanticity of three novel adjuvants including TLR4 (glucopyranosyl lipid adjuvant-squalene emulsion), TLR9 (IC31®), and Mincle (CAF01) agonists, which all induce germinal centers (GCs) and potent antibody responses to influenza hemagglutinin (HA) in adult mice. In neonates, a single dose of HA formulated into each adjuvant induced T follicular helper (TFH) cells. However, only HA/CAF01 elicited significantly higher and sustained antibody responses, engaging neonatal B cells to differentiate into GCs already after a single dose. Although antibody titers remained lower than in adults, HA-specific responses induced by a single neonatal dose of HA/CAF01 were sufficient to confer protection against influenza viral challenge. Postulating that the neonatal adjuvanticity of CAF01 may result from the functionality of the C-type lectin receptor (CLR) Mincle in early life we asked whether other C-type lectin agonists would show a similar neonatal adjuvanticity. Replacing the Mincle agonist trehalose 6,6'-dibehenate by Curdlan, which binds to Dectin-1, enhanced antibody responses through the induction of similar levels of TFH, GCs and bone marrow high-affinity plasma cells. Thus, specific requirements of early life B cells may already be met after a single vaccine dose using CLR-activating agonists, identified here as promising B cell immunostimulators for early life vaccines when included into cationic liposomes.


Assuntos
Adjuvantes Imunológicos , Linfócitos B/imunologia , Centro Germinativo/imunologia , Glicolipídeos/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , beta-Glucanas/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Feminino , Glicolipídeos/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Lectinas Tipo C/agonistas , Lectinas Tipo C/metabolismo , Lipossomos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/agonistas , Receptor Toll-Like 9/metabolismo , beta-Glucanas/farmacologia
4.
J. pediatr. (Rio J.) ; 94(1): 23-30, Jan.-Feb. 2018. tab, graf
Artigo em Inglês | LILACS-Express | ID: biblio-894095

RESUMO

Abstract Objective: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. Methods: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n = 249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. Results: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. Conclusions: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.


Resumo Objetivo: Avaliar o papel do raios X de tórax na identificação de casos de pneumonia adquirida na comunidade (PAC) causada por agentes bacterianos. Métodos: A frequência de infecção por Streptococcus pneumoniae, Haemophilus influenzae e Moraxella catarrhalis em crianças com PAC não hospitalizadas foi comparada com a presença de confirmação radiológica da pneumonia (n = 249 crianças com pneumonia radiologicamente confirmada e 366 crianças com raios X de tórax normal). Infecção por S. pneumoniae foi diagnosticada com base na resposta sorológica a pelo menos uma dentre oito proteínas pneumocócicas investigadas (aumento ≥ 2 vezes nos níveis de IgG em relação a Ply, CbpA, PspA1 e 2, PhtD, StkP-C e PcsB-N ou aumento≥ 1,5 vez em relação aPcpA). Infecção por H. influenzae e M. catarrhalis foi definida por aumento ≥ 2 vezes nos níveis de IgG específica a antígenos de cada agente. Resultados: Crianças com pneumonia radiologicamente confirmada apresentaram maior taxa de infecção pelo pneumococo. Além disso, a presença de infecção pneumocócica foi um fator preditor de pneumonia radiologicamente confirmada, o que aumenta sua chance de detecção em 2,8 vezes (IC 95%: 1,8-4,3). O valor preditivo negativo do raios X normal para a infecção por S. pneumoniae foi 86,3% (IC95%: 82,4%-89,7%). Não houve diferença nas frequências de infecção por H. influenzae e M. catarrhalis entre crianças com PAC com ou sem confirmação radiológica. Conclusão: Crianças com diagnóstico clínico de PAC submetidas a um raios X de tórax que apresentam confirmação radiológica têm maior taxa de infecção por S. pneumoniae comparadas com as crianças com raios X normal.

5.
J Pediatr (Rio J) ; 94(1): 23-30, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28668258

RESUMO

OBJECTIVE: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. METHODS: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n=249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. RESULTS: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. CONCLUSIONS: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.


Assuntos
Infecções por Haemophilus/diagnóstico por imagem , Infecções por Moraxellaceae/diagnóstico por imagem , Pneumonia Bacteriana/diagnóstico por imagem , Pneumonia Bacteriana/microbiologia , Radiografia Torácica , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/sangue , Pré-Escolar , Infecções Comunitárias Adquiridas/diagnóstico por imagem , Infecções Comunitárias Adquiridas/microbiologia , Feminino , Haemophilus influenzae/imunologia , Haemophilus influenzae/isolamento & purificação , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lactente , Masculino , /isolamento & purificação , Pneumonia Pneumocócica/diagnóstico por imagem , Estudos Prospectivos , Sensibilidade e Especificidade , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/isolamento & purificação
6.
PLoS One ; 12(9): e0184357, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28863166

RESUMO

We have previously shown that the Outer surface protein A (OspA) based Lyme borreliosis vaccine VLA15 induces protective immunity in mice. Herein, we report the induction of protective immunity by VLA15 with mouse models using ticks infected with B. burgdorferi (OspA serotype 1), B. afzelii (OspA serotype 2) and B. bavariensis (OspA serotype 4) or with in vitro grown B. garinii (OspA serotype 5 and 6) for challenge. For B. garinii (OspA serotype 3), we have developed a growth inhibition assay using chicken complement and functional antibodies targeting B. garinii (OspA serotype 3) could be demonstrated after immunization with VLA15. Furthermore, following three priming immunizations, a booster dose was administered five months later and the induction of immunological memory could be confirmed. Thus, the antibody titers after the booster dose were increased considerably compared to those after primary immunization. In addition, the half-lives of anti-OspA serotype specific antibodies after administration of the booster immunization were longer than after primary immunization. Taken together, we could show that VLA15 induced protection in mice against challenge with four different clinically relevant Borrelia species (B. burgdorferi, B. afzelii, B. garinii and B. bavariensis) expressing five of the six OspA serotypes included in the vaccine. The protection data is supported by functional assays showing efficacy against spirochetes expressing any of the six OspA serotypes (1 to 6). To our knowledge, this is the first time a Lyme borreliosis vaccine has been able to demonstrate such broad protection in preclinical studies. These new data provide further promise for the clinical development of VLA15 and supports our efforts to provide a new Lyme borreliosis vaccine available for global use.


Assuntos
Antígenos de Superfície/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Grupo Borrelia Burgdorferi/genética , Lipoproteínas/genética , Vacinas contra Doença de Lyme/imunologia , Doença de Lyme/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Sorogrupo
7.
JCI Insight ; 2(6): e83527, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28352649

RESUMO

Chikungunya virus (CHIKV) is rapidly spreading across the globe, and millions are infected. Morbidity due to this virus is a serious threat to public health, but at present, there is no vaccine against this debilitating disease. We have recently developed a number of vaccine candidates, and here we have evaluated 3 of them in a nonhuman primate model. A single immunization with an attenuated strain of CHIKV (Δ5nsP3), a homologous prime-boost immunization with a DNA-launched RNA replicon encoding CHIKV envelope proteins (DREP-E), and a DREP-E prime followed by a recombinant modified vaccinia virus Ankara encoding CHIKV capsid and envelope (MVA-CE) boost all induced protection against WT CHIKV infection. The attenuated Δ5nsP3 virus proved to be safe and did not show any clinical signs typically associated with WT CHIKV infections such as fever, skin rash, lymphopenia, or joint swelling. These vaccines are based on an East/Central/South African strain of Indian Ocean lineage, but they also generated neutralizing antibodies against an isolate of the Asian genotype that now is rapidly spreading across the Americas. These results form the basis for clinical development of an efficacious CHIKV vaccine that generates both humoral and cellular immunity with long-term immunological memory.


Assuntos
Febre de Chikungunya/prevenção & controle , Vírus Chikungunya/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Macaca fascicularis , Vacinas Atenuadas/efeitos adversos , Vacinas Virais/efeitos adversos
9.
Sci Rep ; 6: 39097, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27958370

RESUMO

A better understanding of the mechanisms of action of human adjuvants could inform a rational development of next generation vaccines for human use. Here, we exploited a genome wide transcriptomics analysis combined with a systems biology approach to determine the molecular signatures induced by four clinically tested vaccine adjuvants, namely CAF01, IC31, GLA-SE and Alum in mice. We report signature molecules, pathways, gene modules and networks, which are shared by or otherwise exclusive to these clinical-grade adjuvants in whole blood and draining lymph nodes of mice. Intriguingly, co-expression analysis revealed blood gene modules highly enriched for molecules with documented roles in T follicular helper (TFH) and germinal center (GC) responses. We could show that all adjuvants enhanced, although with different magnitude and kinetics, TFH and GC B cell responses in draining lymph nodes. These results represent, to our knowledge, the first comparative systems analysis of clinically tested vaccine adjuvants that may provide new insights into the mechanisms of action of human adjuvants.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Redes Reguladoras de Genes/efeitos dos fármacos , Centro Germinativo/imunologia , Glucosídeos/administração & dosagem , Lipídeo A/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Oligopeptídeos/administração & dosagem , Linfócitos T Auxiliares-Indutores/imunologia , Imunidade Adaptativa , Adjuvantes Imunológicos/farmacologia , Animais , Combinação de Medicamentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Centro Germinativo/efeitos dos fármacos , Glucosídeos/farmacologia , Humanos , Lipídeo A/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Oligopeptídeos/farmacologia , Análise de Sistemas , Biologia de Sistemas , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Vacinação
10.
J Proteome Res ; 15(9): 3055-97, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27403532

RESUMO

Moraxella catarrhalis, a Gram-negative bacterium, is an important respiratory pathogen causing acute otitis media and exacerbations of chronic obstructive pulmonary disease. Adhesion of the pathogen to human epithelial cells is mediated via bacterial membrane adhesin proteins. To identify the surface proteome of Moraxella catarrhalis, we applied different membrane protein extraction methods in combination with different proteomic technologies. Proteins from preparations of outer membrane vesicles and from carbonate extractions were analyzed using either a gel-based nano-HPLC-MS/MS technique or 2D-LC-MS/MS. Furthermore, because glycosaminoglycans (GAGs) play an important role for microbial entry into human cells, the GAG-binding membranome of Moraxella catarrhalis was investigated using a glycan-based pull-down approach. By these means, potential vaccine protein candidates that were previously selected by the ANTIGENome technology were confirmed, but importantly also novel proteins were identified as candidates.


Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Proteoma/análise , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Infecções por Moraxellaceae/prevenção & controle , Infecções por Moraxellaceae/terapia , Ligação Proteica , Proteômica/métodos
11.
J Immunol Methods ; 433: 31-7, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26928648

RESUMO

The etiological diagnosis of infection by Streptococcus pneumoniae in children is difficult, and the use of indirect techniques is frequently warranted. We aimed to study the use of pneumococcal proteins for the serological diagnosis of pneumococcal infection in children with pneumonia. We analyzed paired serum samples from 13 Brazilian children with invasive pneumococcal pneumonia (positive control group) and 23 Finnish children with viral pharyngitis (negative control group), all aged <5years-old. Children with pharyngitis were evaluated for oropharyngeal colonization, and none of them carried S. pneumoniae. We used a multiplex bead-based assay with eight proteins: Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP and PcsB. The optimal cut-off for increase in antibody level for the diagnosis of pneumococcal infection was determined for each antigen by ROC curve analysis. The positive control group had a significantly higher rate of ≥2-fold rise in antibody levels against all pneumococcal proteins, except Ply, compared to the negative controls. The cut-off of ≥2-fold increase in antibody levels was accurate for pneumococcal infection diagnosis for all investigated antigens. However, there was a substantial increase in the accuracy of the test with a cut-off of ≥1.52-fold rise in antibody levels for PcpA. When using the investigated protein antigens for the diagnosis of pneumococcal infection, the detection of response against at least one antigen was highly sensitive (92.31%) and specific (91.30%). The use of serology with pneumococcal proteins is a promising method for the diagnosis of pneumococcal infection in children with pneumonia. The use of a ≥2-fold increase cut-off is adequate for most pneumococcal proteins.


Assuntos
Anticorpos Antibacterianos/sangue , Imunoensaio/métodos , Pneumonia Pneumocócica/diagnóstico , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Brasil , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Microesferas , Estudos Prospectivos , Curva ROC , Sensibilidade e Especificidade , Testes Sorológicos , Streptococcus pneumoniae/isolamento & purificação
12.
Pediatr Infect Dis J ; 35(6): 683-9, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26954601

RESUMO

BACKGROUND: Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are common causative agents of respiratory infections. Pneumococcal conjugate vaccines have been introduced recently, but their effect on the natural immunity against protein antigens from these pathogens has not been elucidated. METHODS: This was an age-matched observational controlled study that evaluated the influence of 10-valent pneumococcal conjugate vaccines on the levels of antibodies and frequencies of antibody responses against proteins from S. pneumoniae, H. influenzae and M. catarrhalis in serum samples of children with community-acquired pneumonia. Eight pneumococcal proteins (pneumolysin, choline-binding protein A, pneumococcal surface protein A families 1 and 2, pneumococcal choline-binding protein A, pneumococcal histidine triad protein D, serine/threonine protein kinase, protein required for cell wall separation of group B streptococcus), 3 proteins from H. influenzae (including protein D) and 5 M. catarrhalis proteins were investigated. RESULTS: The study group comprised 38 vaccinated children and 114 age-matched controls (median age: 14.5 vs. 14.6 months, respectively; P = 0.997), all with community-acquired pneumonia. There was no difference on clinical baseline characteristics between vaccinated and unvaccinated children. Vaccinated children had significantly lower levels of antibodies against 4 of the studied pneumococcal antigens (P = 0.048 for Ply, P = 0.018 for pneumococcal surface protein A, P = 0.001 for StkP and P = 0.028 for PcsB) and higher levels of antibodies against M. catarrhalis (P = 0.015). Nevertheless, the vaccination status did not significantly affect the rates of antibody responses against S. pneumoniae, H. influenzae and M. catarrhalis. CONCLUSIONS: In spite of the differences that have been found on the level of natural antibodies, no effect from pneumococcal vaccination was observed on the rate of immune responses associated with community-acquired pneumonia against protein antigens from S. pneumoniae, H. influenzae and M. catarrhalis.


Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Haemophilus influenzae/imunologia , Vacinas Pneumocócicas/imunologia , Pneumonia Bacteriana/microbiologia , Streptococcus pneumoniae/imunologia , Formação de Anticorpos , Proteínas de Bactérias/imunologia , Pré-Escolar , Infecções Comunitárias Adquiridas/microbiologia , Feminino , Haemophilus influenzae/isolamento & purificação , Humanos , Lactente , Masculino , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/isolamento & purificação
13.
Sci Rep ; 6: 19570, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26791076

RESUMO

The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.


Assuntos
Adjuvantes Imunológicos , Antígenos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Vacinas/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/prevenção & controle , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imunidade Celular , Imunidade Humoral , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinação
14.
Vaccine ; 33(44): 5982-8, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26277070

RESUMO

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.


Assuntos
Antígenos de Superfície/administração & dosagem , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Borrelia/imunologia , Lipoproteínas/administração & dosagem , Lipoproteínas/imunologia , Doença de Lyme/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Vacinas Bacterianas/química , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/isolamento & purificação , Doença de Lyme/imunologia , Camundongos Endogâmicos C3H , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação
15.
Vet Immunol Immunopathol ; 166(1-2): 8-21, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26004943

RESUMO

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of midges (Culicoides spp.). IgE-mediated reactions are often involved in the pathogenesis of this disease. IBH does not occur in Iceland due to the absence of Culicoides, but it occurs with a high frequency in Icelandic horses exported to mainland Europe, where Culicoides are present. We hypothesize that immunization with the Culicoides allergens before export could reduce the incidence of IBH in exported Icelandic horses. The aim of the present study was therefore to compare intradermal and intralymphatic vaccination using four purified recombinant allergens, in combination with a Th1 focusing adjuvant. Twelve horses were vaccinated three times with 10µg of each of the four recombinant Culicoides nubeculosus allergens. Six horses were injected intralymphatically, three with and three without IC31(®), and six were injected intradermally, in the presence or absence of IC31(®). Antibody responses were measured by immunoblots and ELISA, potential sensitization in a sulfidoleukotriene release test and an intradermal test, cytokine and FoxP3 expression with real time PCR following in vitro stimulation of PBMC. Immunization with the r-allergens induced a significant increase in levels of r-allergen-specific IgG1, IgG1/3, IgG4/7, IgG5 and IgG(T). Application of the r-allergens in IC31(®) adjuvant resulted in a significantly higher IgG1, IgG1/3, IgG4/7 allergen-specific response. Intralymphatic injection was slightly more efficient than intradermal injection, but the difference did not reach significance. Testing of the blocking activity of the sera from the horses immunized intralymphatically with IC31(®) showed that the generated IgG antibodies were able to partly block binding of serum IgE from an IBH-affected horse to these r-allergens. Furthermore, IgG antibodies bound to protein bands on blots of C. nubeculosus salivary gland extract. No allergen-specific IgE was induced and there was no indication of induction of IgE-mediated reactions, as horses neither responded to Culicoides extract stimulation in a sulfidoleukotriene release test, nor developed a relevant immediate hypersensitivity reaction to the recombinant allergens in skin test. IL-4 expression was significantly higher in horses vaccinated intralymphatically without IC31(®), as compared to horses intradermally vaccinated with IC31(®). Both routes gave higher IL-10 expression with IC31(®). Both intralymphatic and intradermal vaccination of horses with recombinant allergens in IC31(®) adjuvant induced an immune response without adverse effects and without IgE production. The horses were not sensitized and produced IgG that could inhibit allergen-specific IgE binding. We therefore conclude that both the injection routes and the IC31(®) adjuvant are strong candidates for further development of immunoprophylaxis and therapy in horses.


Assuntos
Alérgenos/imunologia , Hipersensibilidade/prevenção & controle , Imunização , Mordeduras e Picadas de Insetos/imunologia , Animais , Combinação de Medicamentos , Cavalos , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Oligodesoxirribonucleotídeos/farmacologia , Oligopeptídeos/farmacologia , Projetos Piloto , Proteínas Recombinantes/imunologia
16.
PLoS One ; 10(3): e0120548, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25798594

RESUMO

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.


Assuntos
Grupo Borrelia Burgdorferi/genética , Genômica , Sequência de Bases , Grupo Borrelia Burgdorferi/isolamento & purificação , Grupo Borrelia Burgdorferi/patogenicidade , Grupo Borrelia Burgdorferi/virologia , Cromossomos Bacterianos/genética , DNA Viral/genética , Loci Gênicos/genética , Genoma Bacteriano/genética , Fases de Leitura Aberta/genética , Filogenia , Plasmídeos/genética , Prófagos/genética , Prófagos/fisiologia , RNA não Traduzido/genética , Análise de Sequência , Especificidade da Espécie , Sequências de Repetição em Tandem/genética , Telômero/genética
17.
MAbs ; 6(6): 1608-20, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25484038

RESUMO

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Autoanticorpos/imunologia , Interleucina-17/imunologia , Timoma/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Afinidade de Anticorpos/imunologia , Autoanticorpos/genética , Autoanticorpos/isolamento & purificação , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interleucina-17/genética , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Timoma/sangue
18.
PLoS One ; 9(11): e113294, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25409015

RESUMO

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.


Assuntos
Proteínas da Membrana Bacteriana Externa/síntese química , Vacinas Bacterianas/síntese química , Borrelia/imunologia , Lipoproteínas/síntese química , Vacinas contra Doença de Lyme/síntese química , Doença de Lyme/prevenção & controle , Animais , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Borrelia/efeitos dos fármacos , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Humanos , Lipoproteínas/química , Lipoproteínas/imunologia , Doença de Lyme/imunologia , Vacinas contra Doença de Lyme/administração & dosagem , Camundongos , Camundongos Endogâmicos C3H , Carrapatos/microbiologia
19.
J Immunol Methods ; 405: 130-43, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24530690

RESUMO

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are pathogens commonly associated with infectious diseases in childhood. This study aimed to develop a fluorescent multiplexed bead-based immunoassay (FMIA) using recombinant proteins for the quantitation of serum IgG antibodies against these bacteria. Eight pneumococcal proteins (Ply, CbpA, PspA1, PspA2, PcpA, PhtD, SP1732-3 and SP2216-1), 3 proteins of H. influenzae (NTHi Protein D, NTHi0371-1, NTHi0830), and 5 proteins of M. catarrhalis (MC Omp CD, MC_RH4_2506, MC_RH4_1701, MC_RH4_3729-1, MC_RH4_4730) were used to develop the FMIA. Optimal coupling concentrations for each protein, comparison of singleplex and multiplex assays, specificity, reproducibility, and correlation to ELISA for six pneumococcal antigens were determined for validation. FMIA was then used to analyze acute and convalescent paired serum samples of 50 children with non-severe pneumonia. The coupling concentrations varied for different antigens, ranging from 1.6 to 32µg of protein/million beads. Correlation between singleplexed and multiplexed assays was excellent, with R≥0.987. The FMIA was specific, reaching >92% homologous inhibition for all specificities; heterologous inhibition ≥20% was found only in six cases. The assay was repeatable, with averages of intra-assay variation ≤10.5%, day-to-day variation ≤9.7% and variation between technicians ≤9.1%. Comparison with ELISA for pneumococcal antigens demonstrated good correlation with R ranging from 0.854 (PspA2) to 0.976 (PcpA). The samples from children showed a wide range of antibody concentrations and increases in convalescent samples. In conclusion, the FMIA was sensitive, specific, and repeatable, using small amounts of recombinant proteins and sera to detect antibodies against S. pneumoniae, H. influenzae and M. catarrhalis. The methodology would be suitable for studies investigating etiological diagnosis and in experimental vaccine studies.


Assuntos
Proteínas de Bactérias/imunologia , Haemophilus influenzae/imunologia , Imunoensaio/métodos , Imunoglobulina G/imunologia , Streptococcus pneumoniae/imunologia , Doença Aguda , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/sangue , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/imunologia , Fluorescência , Humanos , Imunoglobulina G/sangue , Lactente , Microesferas , Otite Média/sangue , Otite Média/diagnóstico , Otite Média/microbiologia , Pneumonia/sangue , Pneumonia/diagnóstico , Pneumonia/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
20.
Clin Vaccine Immunol ; 21(2): 253-5, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24334688

RESUMO

Dry tetanus toxoid (TTx) patches were formulated without any adjuvant, with excipients to impart antigen stabilization and to enhance skin delivery. The booster effects of the TTx patches were assessed using a guinea pig model. The study revealed significant rises in TTx IgG titers induced by the TTx patches after a low-dose subcutaneous (s.c.) prime with TTx adsorbed to aluminum hydroxide. The TTx patch can therefore be considered an effective alternative to a subcutaneous booster.


Assuntos
Imunização Secundária/métodos , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/imunologia , Tétano/prevenção & controle , Administração Cutânea , Animais , Anticorpos Antibacterianos/sangue , Antitoxinas/sangue , Feminino , Cobaias , Imunoglobulina G/sangue
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