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1.
Sci Rep ; 11(1): 21849, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34750472

RESUMO

The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.


Assuntos
Adjuvantes Imunológicos/química , Antígenos Arqueais/química , Vacinas contra COVID-19/uso terapêutico , Lipídeos/química , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Peso Corporal , COVID-19/terapia , Chlorocebus aethiops , Cricetinae , Citocinas/metabolismo , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Passiva , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Peptídeos/química , Domínios Proteicos , SARS-CoV-2 , Receptores Toll-Like/imunologia , Células Vero , Carga Viral
2.
J Liposome Res ; 31(3): 237-245, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32583693

RESUMO

Archaeosomes, composed of sulphated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. In addition to efficacy, the stability of vaccine components including the adjuvant is an important parameter to consider when developing novel vaccine formulations. To properly evaluate the potential of SLA glycolipids to be used as vaccine adjuvants in a clinical setting, a comprehensive evaluation of their stability is required. Herein, we evaluated the long term stability of preformed empty SLA archaeosomes prior to admixing with antigen at 4 °C or 37 °C for up to 6 months. In addition, the stability of adjuvant and antigen was evaluated for up to 1 month following admixing. Multiple analytical parameters evaluating the molecular integrity of SLA and the liposomal profile were assessed. Following incubation at 4 °C or 37 °C, the SLA glycolipid did not show any pattern of degradation as determined by mass spectroscopy, nuclear magnetic resonance (NMR) and thin layer chromatography (TLC). In addition, SLA archaeosome vesicle characteristics, such as size, zeta potential, membrane fluidity and vesicular morphology, were largely consistent throughout the course of the study. Importantly, following storage for 6 months at both 4 °C and 37 °C, the adjuvant properties of empty SLA archaeosomes were unchanged, and following admixing with antigen, the immunogenicity of the vaccine formulations was also unchanged when stored at both 4 °C and 37 °C for up to 1 month. Overall this indicates that SLA archaeosomes are highly stable adjuvants that retain their activity over an extended period of time even when stored at high temperatures.


Assuntos
Lipossomos , Vacinas , Antígenos Arqueais , Imunidade Celular , Lipídeos
3.
Front Immunol ; 11: 605288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33304354

RESUMO

Schistosomiasis threatens 800 million people worldwide. Chronic pathology manifests as hepatosplenomegaly, and intestinal schistosomiasis caused by Schistosoma mansoni can lead to liver fibrosis, cirrhosis, and blood in the stool. To assist the only FDA-approved drug, praziquantel, in parasite elimination, the development of a vaccine would be of high value. S. mansoni Cathepsin B (SmCB) is a well-documented vaccine target for intestinal schistosomiasis. Herein, we test the increased efficacy and immunogenicity of SmCB when combined with sulfated lactosyl archaeol (SLA) archaeosomes or AddaVax™ (a squalene based oil-in-water emulsion). Both vaccine formulations resulted in robust humoral and cell mediated immune responses. Impressively, both formulations were able to reduce parasite burden greater than 40% (WHO standard), with AddaVax™ reaching 86.8%. Additionally, SmCB with both adjuvants were able to reduce granuloma size and the amount of larval parasite hatched from feces, which would reduce transmission. Our data support SmCB as a target for S. mansoni vaccination; especially when used in an adjuvanted formulation.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos Arqueais/farmacologia , Catepsina B/farmacologia , Proteínas de Helminto/farmacologia , Lipídeos/farmacologia , Polissorbatos/farmacologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Esqualeno/farmacologia , Vacinas Sintéticas/farmacologia , Animais , Anticorpos/sangue , Catepsina B/imunologia , Células Cultivadas , Citocinas/metabolismo , Composição de Medicamentos , Feminino , Proteínas de Helminto/imunologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunização , Imunogenicidade da Vacina , Camundongos Endogâmicos C57BL , Contagem de Ovos de Parasitas , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Caramujos , Vacinas Sintéticas/imunologia
4.
Int J Pharm ; 561: 187-196, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30836154

RESUMO

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that can be used as adjuvants to induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigen. However, the entrapment efficiency of antigen within archaeosomes constituted using standard liposome forming methodology is often only 5-40%. In this study, we evaluated different formulation methods using a simple semi-synthetic archaeal lipid (SLA, sulfated lactosyl archaeol) and two different antigens, ovalbumin (OVA) and hepatitis B surface antigen (HBsAg). Antigen was entrapped within archaeosomes using the conventional thin film hydration-rehydration method with or without removal of non-entrapped antigen, or pre-formed empty archaeosomes were simply admixed with an antigen solution. Physicochemical characteristics were determined (size distribution, zeta potential, vesicle morphology and lamellarity), as well as location of antigen relative to bilayer using cryogenic transmission electron microscopy (TEM). We demonstrate that antigen (OVA or HBsAg) formulated with SLA lipid adjuvants using all the different methodologies resulted in a strong antigen-specific immune response. Nevertheless, the advantage of using a drug substance process that comprises of simply admixing antigen with pre-formed empty archaeosomes, represents a simple, efficient and antigenic dose-sparing formulation for adjuvanting and delivering vaccine antigens.


Assuntos
Adjuvantes Imunológicos/química , Antígenos Arqueais/imunologia , Archaea/imunologia , Portadores de Fármacos/química , Lipídeos/química , Lipossomos/química , Vacinas/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos/sangue , Contagem de Células , Fenômenos Químicos , Feminino , Antígenos de Superfície da Hepatite B/imunologia , Imunidade Celular/efeitos dos fármacos , Interferon gama/metabolismo , Lipossomos/ultraestrutura , Camundongos , Ovalbumina/imunologia , Baço/metabolismo , Vacinas/química
5.
Environ Microbiol ; 14(9): 2495-510, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22564293

RESUMO

In this study we examined the microbial community composition and operating metabolisms on the surface and in the permafrost of Deception Island, (Antarctica) with an on site antibody microarray biosensor. Samples (down to a depth of 4.2 m) were analysed with LDChip300 (Life Detector Chip), an immunosensor containing more than 300 antibodies targeted to bacterial and archaeal antigens. The immunograms showed positive antigen-antibody reactions in all surface samples (lichens, pyroclasts) and the top layer of the permafrost. The results indicated the presence of exopolysaccharides, bacteria belonging to the Alpha-, Delta- and Gammaproteobacteria, Bacteroidetes, Gram-positive Actinobacteria and Firmicutes, as well as archaeal species, most probably Methanobacterium spp. Positive reactions with antibodies to proteins and peptides revealed the presence of nitrogen fixation (NifHD, GlnB, HscA), methanogenic (McrB), iron homeostasis and iron scavenging (ferritins and DPS proteins) proteins, as well as ABC transporters, which indicated that these processes were operating at the time of sampling. These results were validated with other molecular ecology techniques such as oligonucleotide microarrays, 16S bacterial rRNA gene sequence analysis, aerobic viable counts and microscopy. Molecular ecology results showed a differentiated pattern along the depth of the drill, being the top active layer the most diverse, with Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes and the phototrophs Cyanobacteria and Chloroflexi as dominant groups. Actinobacteria and Firmicutes were dominant in depths from 0.5 to 2 m, and Betaproteobacteria from 3 to 4.2 m. The geochemical analysis revealed the presence of low molecular weight organic acids (acetate, formate) which could be used by microorganisms as energy sources for sulfate, nitrate and metal reduction under anaerobic conditions.


Assuntos
Archaea , Bactérias , Biodiversidade , Ilhas , Microbiologia do Solo , Regiões Antárticas , Antígenos Arqueais/metabolismo , Antígenos de Bactérias/metabolismo , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Carga Bacteriana , Biomarcadores/análise , Microscopia Eletrônica de Varredura , Filogenia , RNA Ribossômico 16S/genética , Solo/química
6.
Mol Oral Microbiol ; 25(2): 112-22, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20331799

RESUMO

Methanobrevibacter oralis is an archaeal species frequently isolated from sites of severe periodontitis. However, its pathogenic roles remain unclear. Here, we aimed to isolate group II chaperonin from M. oralis and examine its antigenicity. The genes encoding two chaperonin subunits (Cpn-1 and Cpn-2) were cloned from M. oralis using polymerase chain reaction and genome walking procedures. Recombinant proteins Cpn-1 and Cpn-2 were generated, and the reactivities of sera from patients with periodontitis were examined by Western immunoblotting. The open reading frames of Cpn-1 and Cpn-2 genes consisted of 1641 and 1614 base pairs, respectively. Putative ATP-binding domains conserved among the chaperonin family were observed in both genes. The deduced amino acid sequences of the two genes showed 28.8-40.0% identity to each of the subunits of human CCT (CCT1-8). Thirty and 29 of 36 patients' sera reacted with the recombinant Cpn-1 and recombinant Cpn-2, respectively. Western immunoblotting using antiserum against human CCT subunits indicated that anti-CCT3 and anti-CCT8 antibodies recognized recombinant Cpn-1. In addition, anti-CCT1, CCT3, CCT6, and CCT8 antibodies recognized an antigen of approximately 60 kDa in M. oralis. The results suggested that the chaperonin subunits of M. oralis were antigenic molecules that were recognized by periodontitis patients and that may cross-react with human chaperonin CCT.


Assuntos
Antígenos Arqueais/imunologia , Chaperoninas do Grupo II/imunologia , Methanobrevibacter/patogenicidade , Periodontite/imunologia , Periodontite/microbiologia , Antígenos Arqueais/genética , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/imunologia , Passeio de Cromossomo , Sequência Conservada/imunologia , Reações Cruzadas , DNA Arqueal/análise , Chaperoninas do Grupo II/genética , Interações Hospedeiro-Patógeno , Humanos , Methanobrevibacter/imunologia , Periodontite/sangue , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA
7.
Biochem Soc Trans ; 32(Pt 2): 184-7, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15046568

RESUMO

This minireview summarizes our current knowledge about archaeal genetic elements in the hyperthermophilic order Thermococcales in the phylum Euryarchaeota. This includes recent work on the first virus of Pyrococcus, PAV1, the discovery of novel unique virus morphotypes in hot deep-sea environments, and preliminary observations on novel cryptic plasmids. We also review the work accomplished over the last 5 years in the development of genetic tools for members of the Pyrococcus and Thermococcus genera, mainly in our laboratories.


Assuntos
Genoma Arqueal , Pyrococcus/genética , Thermococcales/genética , Thermococcus/genética , Antígenos Arqueais/química , DNA Arqueal/ultraestrutura , Vetores Genéticos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Compostos Organometálicos/farmacologia , Plasmídeos/metabolismo , Transgenes , Vírus/genética
8.
Int J Syst Evol Microbiol ; 50 Pt 1: 43-53, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10826786

RESUMO

Using a combination of 16S rRNA analysis and antigenic fingerprinting consisting of new and published data, the phylogenetic position of 18 thermophilic isolates currently classified as Methanobacterium species was reinvestigated. The results were verified by independent methods, including, where applicable, plasmid and phage typing. Comparative analysis of 16S rRNA data for 30 strains belonging to the order Methanobacteriales strongly suggested that mesophilic and thermophilic Methanobacterium isolates are distantly related and should be assigned to separate genera. For the thermophilic strains the genus Methanothermobacter was initially proposed by Boone, Whitman and Rouvière. Furthermore, the results support a reclassification of 15 isolates in three species within the proposed genus: (i) Methanothermobacter thermautotrophicus comb. nov., containing eight isolates, six of which are able to utilize formate (type strain deltaHT); (ii) Methanothermobacter wolfeii comb. nov., containing four formate-utilizing isolates (type strain DSM 2970T); (iii) Methanothermobacter marburgensis sp. nov., containing three obligately autotrophic isolates (type strain MarburgT). Of the nine isolates formerly referred to as Methanobacterium thermoformicicum, six were reclassified as Methanothermobacter thermautotrophicus and three as Methanothermobacter wolfeii.


Assuntos
Methanobacteriaceae/classificação , Methanobacteriaceae/genética , Methanobacterium/classificação , Methanobacterium/genética , Antígenos Arqueais/classificação , Tipagem de Bacteriófagos , Sequência de Bases , DNA Arqueal/genética , DNA Ribossômico/genética , Methanobacteriaceae/imunologia , Methanobacteriaceae/fisiologia , Methanobacterium/imunologia , Methanobacterium/fisiologia , Dados de Sequência Molecular , Fenótipo , Filogenia , Plasmídeos , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Temperatura
9.
Extremophiles ; 3(4): 239-45, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10591013

RESUMO

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates.


Assuntos
Antígenos Arqueais/análise , Thermococcales/classificação , Thermococcus/classificação , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Água do Mar/microbiologia , Thermococcales/imunologia , Thermococcales/ultraestrutura , Thermococcus/imunologia , Thermococcus/ultraestrutura
10.
Appl Environ Microbiol ; 57(6): 1728-34, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16348508

RESUMO

Upflow anaerobic sludge blanket bioreactor granules were used as an experimental model microbial consortium to study the dynamics and distribution of methanogens. Immunologic methods revealed a considerable diversity of methanogens that was greater in mesophilic granules than in the same granules 4 months after a temperature shift from 38 to 55 degrees C. During this period, the sizes of the methanogenic subpopulations changed with distinctive profiles after the initial reduction caused by the shift. Methanogens antigenically related to Methanobrevibacter smithii PS and ALI, Methanobacterium hungatei JF1, and Methanosarcina thermophila TM1 increased rapidly, reached a short plateau, and then fell to lower concentrations that persisted for the duration of the experiment. A methanogen related to Methanogenium cariaci JR1 followed a similar profile at the beginning, but it soon diminished below detection levels. Methanothrix rods weakly related to the strain Opfikon increased rapidly, reaching a high-level, long-lasting plateau. Two methanogens related to Methanobrevibacter arboriphilus AZ and Methanobacterium thermoautotrophicum DeltaH emerged from very low levels before the temperature shift and multiplied to attain their highest numbers 4 months after the shift. Histochemistry and immunohistochemistry revealed thick layers, globular clusters, and lawns of variable density which were distinctive of the methanogens related to M. thermoautotrophicum DeltaH, M. thermophila TM1, and M. arboriphilus AZ and M. soehngenii Opfikon, respectively, in thin sections of granules grown at 55 degrees C for 4 months. Mesophilic granules showed a different pattern of methanogenic subpopulations.


Assuntos
Biodiversidade , Reatores Biológicos/microbiologia , Euryarchaeota/fisiologia , Esgotos/microbiologia , Anticorpos Antiarchaea/metabolismo , Antígenos Arqueais/análise , Contagem de Colônia Microbiana
11.
Appl Environ Microbiol ; 55(8): 1996-2001, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16347990

RESUMO

A fixed-bed loop, high-rate anaerobic bioreactor treating sulfite evaporator condensate was sampled when it reached steady state and afterwards following perturbations during a 14-month period. By using immunotechnology, it was observed that shifts in methanogenic subpopulations occurred in association with perturbations, such as restarting and relocating the biomass into a different tank. Methanogens related to Methanobacterium bryantii MoHG and Methanobrevibacter smithii ALI were numerous throughout the observation period, while Methanosarcina mazei S6 and Methanosarcina thermophila TM1 were found in the early and late samples, respectively. Also, Methanobacterium formicicum was more numerous at the top portion of the bioreactor, while Methanobrevibacter arboriphilus AZ and DC were at the bottom. Sample formalinization required for prolonged storage proved suitable for antigen preservation.


Assuntos
Biodiversidade , Reatores Biológicos/microbiologia , Euryarchaeota/fisiologia , Anticorpos Antiarchaea/metabolismo , Antígenos Arqueais/análise , Euryarchaeota/isolamento & purificação , Formaldeído/química , Técnicas Microbiológicas/métodos , Estresse Fisiológico/fisiologia , Sulfitos/química , Fatores de Tempo , Eliminação de Resíduos Líquidos/métodos
12.
Appl Environ Microbiol ; 54(3): 693-8, 1988 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16347581

RESUMO

The methanogenic flora from two types of turbulent, high-rate reactors was studied by immunologic methods as well as by phase-contrast, fluorescence, and scanning electron microscopy. The reactors were a fluidized sand-bed biofilm ANITRON reactor and an ultrafiltration membrane-associated suspended growth MARS reactor (both trademarks of Air Products and Chemicals, Inc., Allentown, Pa.). Conventional microscopic methods revealed complex mixtures of microbes of a range of sizes and shapes, among which morphotypes resembling Methanothrix spp. and Methanosarcina spp. were noticed. Precise identification of these and other methanogens was accomplished by antigenic fingerprinting with a comprehensive panel of calibrated antibody probes of predefined specificity spectra. The methanogens identified showed morphotypes and antigenic fingerprints indicating their close similarity with the following reference organisms: Methanobacterium formicicum MF and Methanosarcina barkeri W in the ANITRON reactor only; Methanosarcina barkeri R1M3, M. mazei S6, Methanogenium cariaci JR1, and Methanobrevibacter arboriphilus AZ in the MARS reactor only; and Methanobrevibacter smithii ALI and Methanothrix soehngenii Opfikon in both reactors. Species diversity and distribution appeared to be, at least in part, dependent on the degree of turbulence inside the reactor.


Assuntos
Reatores Biológicos/microbiologia , Euryarchaeota/fisiologia , Anaerobiose/fisiologia , Anticorpos Antiarchaea/metabolismo , Antígenos Arqueais/metabolismo , Técnicas de Tipagem Bacteriana , Biodiversidade , Euryarchaeota/classificação , Euryarchaeota/imunologia , Euryarchaeota/isolamento & purificação , Microscopia Eletrônica de Varredura , Eliminação de Resíduos/métodos
13.
Appl Environ Microbiol ; 54(3): 835-6, 1988 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16347591

RESUMO

Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of Methanobacterium formicicum, Methanosarcina barkeri, several different immunotypes of Methanobacterium bryantii, and a coccoid methanogen distinct from the reference immunotypes were identified.


Assuntos
Microbiologia Ambiental , Euryarchaeota/isolamento & purificação , Euryarchaeota/fisiologia , Eliminação de Resíduos , Anticorpos Antiarchaea/metabolismo , Antígenos Arqueais/metabolismo , Técnicas de Tipagem Bacteriana , Biodiversidade , Euryarchaeota/classificação , Euryarchaeota/citologia
14.
Appl Environ Microbiol ; 54(1): 79-86, 1988 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16347541

RESUMO

To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive significance.


Assuntos
Anticorpos Antiarchaea/metabolismo , Biodiversidade , Reatores Biológicos/microbiologia , Euryarchaeota/classificação , Euryarchaeota/imunologia , Eliminação de Resíduos , Antígenos Arqueais/análise , Euryarchaeota/isolamento & purificação , Methanobacterium/isolamento & purificação , Methanobacterium/fisiologia , Methanobrevibacter/isolamento & purificação , Methanobrevibacter/fisiologia , Eliminação de Resíduos/métodos , Especificidade da Espécie
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