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1.
Vaccine ; 32(24): 2904-10, 2014 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-24598720

RESUMO

BACKGROUND: Nasal vaccination is considered to be a promising alternative for parenteral vaccination against influenza virus as it is non-invasive and offers the opportunity to elicit strong antigen-specific responses both systemic and locally at the port of entry of the pathogen. Previous studies showed that non-living bacterium-like particles (BLPs) from the food-grade bacterium Lactococcus lactis are effective stimulators of local and systemic immune responses when administered intranasally. Moreover, in vitro, BLPs specifically interact with human Toll-like receptor 2 (TLR2), suggestive of a role for TLR2 dependent immune activation by BLPs. METHODS: In the present study, we examined the role of TLR2 in vivo in immune activation after nasal administration of BLP mixed with split influenza vaccine (BLP-SV) of influenza A virus (IAV) using TLR2 knockout mice. RESULTS: The systemic Th1 cell and subsequent B-cell responses induced after intranasal BLP-SV vaccination depended on the interaction of BLPs with TLR2. Notably, the BLP-SV-induced class switch to IgG2c depended on the interaction of BLP with TLR2. Local induced IAV-specific Th1 cell responses and the mucosal B-cell responses also depended on interaction of BLP with TLR2. Strongly reduced SIgA levels were observed in TLR2 knockout mice both in the nasal and vaginal lavages. In addition, detailed analysis of the T-cell response revealed that nasal BLP-SV vaccination promoted Th1/Th17 immune responses that coincided with increased IAV-specific IgG2c antibody production. DISCUSSION: Altogether these results indicate that nasal BLP-SV vaccination induces IAV-specific T-cell and B-cell responses, both systemically and at the site of virus entry in a TLR2-dependent manner.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Imunidade nas Mucosas , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Receptor 2 Toll-Like/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Feminino , Imunoglobulina A Secretora/imunologia , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Lactococcus lactis/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th1/imunologia , Receptor 2 Toll-Like/genética , Vacinas de Produtos Inativados/imunologia
2.
Vaccine ; 28(50): 7963-9, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-20946860

RESUMO

Gram-positive enhancer matrix (GEM) particles, produced from non-genetically modified Lactococcus lactis bacteria have an inherent immunostimulatory activity. It was investigated whether co-administration of GEM particles can reduce the amount of influenza subunit vaccine (HA) necessary to protect mice from viral infection. Decreasing HA amounts of 5, 1, 0.2 and 0.04µg admixed with GEM particles were tested in intramuscular immunizations. Combinations of GEM and seasonal HA (A/Wisconsin/67/2005 [H3N2]) induced significantly higher systemic and better Th1/Th2-type balanced immune responses than HA alone. Addition of GEM to 0.04µg HA resulted in similar HI titers as 1-5µg non-adjuvanted HA. To test the protective efficacy of the adjuvanted combination, mice were immunized with influenza subunit vaccine A/PR/8/34 (H1N1) and then challenged with live virus (A/PR/8/34). Mice immunized with 1µg HA+GEM showed undetectable virus titers in the lungs 5 days after challenge, whereas mice immunized with 1µg HA alone showed detectable levels of virus in the lungs. Interestingly, mice vaccinated with the 0.04µg HA+GEM vaccine demonstrated reduced lung virus titers and a reduction in weight that was similar as that in mice vaccinated with 1µg non-adjuvanted HA. These results indicate that the use of GEM as immunostimulant allows for a strong reduction in the antigen dose as compared to the benchmark vaccine by using GEM particles. Thus, addition of GEM can strongly potentiate immunogenicity of influenza subunit vaccine both quantitatively and qualitatively.


Assuntos
Adjuvantes Imunológicos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Relação Dose-Resposta Imunológica , Testes de Hemaglutinação , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Injeções Intramusculares , Lactococcus lactis/química , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Carga Viral
3.
Eur J Pharm Biopharm ; 76(3): 470-4, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20719246

RESUMO

In this study, a liquid formulation of influenza subunit vaccine admixed with Gram-positive enhancer matrix (GEM) particles as adjuvant was delivered to upper and lower parts of intestinal tract. The aim was to determine the most effective immunization site in the intestines. Mice were vaccinated with a liquid formulation of GEM and influenza subunit vaccine orally and rectally. The oral administration of the vaccine with GEM particles induced a better systemic and mucosal immune response than oral (vaccine only) and rectal (with and without adjuvant) immunizations. Rectal administration elicited high IgG1 responses but little IgG2a, indicating a Th2 dominated immune response. In contrast, the oral immunization with GEM particles elicited a balanced IgG1 and IgG2a response. In conclusion, our results demonstrate that GEM-adjuvanted influenza vaccine should be targeted to the upper part of the intestinal tract.


Assuntos
Adjuvantes Imunológicos , Anticorpos Antivirais/sangue , Trato Gastrointestinal/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Lactococcus lactis/imunologia , Administração Oral , Administração Retal , Animais , Imunidade nas Mucosas , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia
4.
Mucosal Immunol ; 3(2): 159-71, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19924118

RESUMO

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated as Gram-positive enhancer matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells (DCs) both in vivo and in vitro. These DCs showed increased capacities for secretion of proinflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4(+) and CD8(+) T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life.


Assuntos
Infecções Bacterianas/prevenção & controle , Lactococcus lactis/imunologia , Células Th1/imunologia , Vacinação , Administração Intranasal , Animais , Animais Recém-Nascidos , Anticorpos/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Camundongos , Mucosa/imunologia , Yersinia pestis/imunologia
5.
J Biol Chem ; 276(27): 25230-5, 2001 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-11309398

RESUMO

The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor importance for cell viability. Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments. Here, we have successfully used molecular phylogeny to predict major and minor SPases. The results were verified with SPases from various bacilli. As predicted, the latter enzymes behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases. Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site. Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase. This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection.


Assuntos
Bacillus/enzimologia , Proteínas de Membrana , Serina Endopeptidases/classificação , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica
6.
J Bacteriol ; 182(20): 5765-70, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11004175

RESUMO

Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.


Assuntos
Bacillus subtilis/enzimologia , Escherichia coli/enzimologia , Proteínas de Membrana , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Primers do DNA , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina Endopeptidases/química
7.
Genes Dev ; 12(15): 2318-31, 1998 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9694797

RESUMO

Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of the sip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sequência Conservada , Primers do DNA/genética , Retículo Endoplasmático/enzimologia , Células Eucarióticas , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Mutação , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/classificação , Serina Endopeptidases/genética
8.
J Biol Chem ; 273(33): 21217-24, 1998 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-9694879

RESUMO

In the present studies, we show that the SecD and SecF equivalents of the Gram-positive bacterium Bacillus subtilis are jointly present in one polypeptide, denoted SecDF, that is required to maintain a high capacity for protein secretion. Unlike the SecD subunit of the pre-protein translocase of Escherichia coli, SecDF of B. subtilis was not required for the release of a mature secretory protein from the membrane, indicating that SecDF is involved in earlier translocation steps. Strains lacking intact SecDF showed a cold-sensitive phenotype, which was exacerbated by high level production of secretory proteins, indicating that protein translocation in B. subtilis is intrinsically cold-sensitive. Comparison with SecD and SecF proteins from other organisms revealed the presence of 10 conserved regions in SecDF, some of which appear to be important for SecDF function. Interestingly, the SecDF protein of B. subtilis has 12 putative transmembrane domains. Thus, SecDF does not only show sequence similarity but also structural similarity to secondary solute transporters. Our data suggest that SecDF of B. subtilis represents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Transcrição Gênica
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