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Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 µg/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r > 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.
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Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/diagnóstico , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Lactente , Pré-Escolar , Adulto , Criança , Adolescente , Pessoa de Meia-Idade , Adulto Jovem , Feminino , Sensibilidade e Especificidade , Antígenos Virais/imunologia , Masculino , Proteínas Virais de Fusão/imunologia , IdosoRESUMO
This is a cross-sectional serosurveillance study for RSV. Between June and September of 2021, a total of 150 sera were collected from 30 individuals in each age group (<5, 5-18, 19-49, 50-64, and ≥65 years). Seroprevalence was estimated using enzyme-linked immunosorbent assays targeting two stabilized prefusion F (preF; DS-Cav1 and SC-TM) and G proteins. The overall seroprevalence was low in young children and older adults, despite them having a higher risk of severe RSV infection. There was a remarkable difference in age-stratified seroprevalence rates between anti-preF and anti-G protein antibodies. Given the high disease burden and low seroprevalence in both infants and old adults, RSV vaccination would be crucial for pregnant women and people aged over 60 years.
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Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-ß (TGF-ß) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-ß reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-ß production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-ß may be an alternative therapy for viral pneumonia.
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Artrite Reumatoide , COVID-19 , Pneumonia Viral , Animais , Camundongos , COVID-19/radioterapia , Inflamação , Citocinas , Dimercaprol , Fatores de Crescimento TransformadoresRESUMO
Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.
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Pneumonia , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Camundongos , Animais , Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vacinas contra Vírus Sincicial Respiratório/química , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Espécies Reativas de Oxigênio , Pulmão , Anticorpos Antivirais , FormaldeídoRESUMO
There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.
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Deinococcus radiodurans is an extremophile, well known to be extremely resistant to external stresses due to its unique physiological system and structure of cellular components. Although the proportion of D. radiodurans has been reported to be negatively correlated with atopic dermatitis, the exact function of D. radiodurans in allergic diseases and its precise mechanisms have not been studied. In the present study, we hypothesize that D. radiodurans or its cellular constituents play a critical role in the skin to prevent allergic inflammatory responses by modulating immunity. Heat-killed D. radiodurans inhibited the production of Th2 cytokines, such as IL-4 and IL-5, induced by ovalbumin (OVA) stimulation in splenocytes from OVA-sensitized mice. Among the cellular constituents of D. radiodurans, such as cell wall (DeinoWall), cell membrane (DeinoMem), and exopolysaccharide (DeinoPol), only DeinoWall inhibited the production of Th2 cytokines and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD), a Th2-predominant allergic disease in mice. Moreover, serum IgE levels and infiltration of mast cells into skin lesions, the markers of Th2 response induced by DNCB application, were significantly inhibited by treatment with DeinoWall. Remarkably, DeinoWall induced the maturation of bone marrow-derived dendritic cells (BMDCs) that promote Th1-biased immunity, which might balance Th1/Th2 and regulate allergic inflammatory responses. Collectively, these results suggest that DeinoWall acts as a major cellular constituent in the negative regulation of allergic inflammatory responses by D. radiodurans and might be a viable candidate for the treatment of allergic diseases.
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Antialérgicos , Deinococcus , Dermatite Atópica , Animais , Antialérgicos/farmacologia , Parede Celular , Citocinas , Deinococcus/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Dinitroclorobenzeno/metabolismo , Imunoglobulina E , Interleucina-4/metabolismo , Interleucina-5 , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/metabolismo , Células Th2RESUMO
Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.
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Melhoramento Biomédico , Vacinas contra Salmonella , Animais , Anticorpos Antibacterianos/genética , Feminino , Humanos , Imunoglobulina G/genética , Camundongos , Mutação , Vacinas contra Salmonella/genética , Salmonella typhimurium , Suínos , Vacinas AtenuadasRESUMO
Initiation and progression of oral infectious diseases are associated with streptococcal species. Bacterial infection induces inflammatory responses together with reactive oxygen species (ROS), often causing cell death and tissue damage in the host. In the present study, we investigated the effects of oral streptococci on cytotoxicity and ROS production in human periodontal ligament (PDL) cells. Streptococcus gordonii showed cell cytotoxicity in a dose- and time-dependent manner. The cytotoxicity might be due to apoptosis since S. gordonii increased annexin V-positive cells, and the cytotoxicity was reduced by an apoptosis inhibitor, Z-VAD-FMK. Other oral streptococci such as Streptococcus mitis, Streptococcus sanguinis, and Streptococcus sobrinus also induced apoptosis, whereas Streptococcus mutans did not. All streptococci tested except S. mutans triggered ROS production in human PDL cells. Interestingly, however, streptococci-induced apoptosis appears to be ROS-independent, as the cell death induced by S. gordonii was not recovered by the ROS inhibitor, resveratrol or n-acetylcysteine. Instead, hydrogen peroxide (H2O2) appears to be important for the cytotoxic effects of streptococci since most oral streptococci except S. mutans generated H2O2, and the cytotoxicity was dramatically reduced by catalase. Furthermore, streptococcal lipoproteins are involved in cytotoxicity, as we observed that cytotoxicity induced by the lipoprotein-deficient S. gordonii mutant was less potent than that by the wild-type and was attenuated by anti-TLR2-neutralizing antibody. Indeed, lipoproteins purified from S. gordonii alone were sufficient to induce cytotoxicity. Notably, S. gordonii lipoproteins did not induce H2O2 or ROS but cooperatively induced cell death when co-treated with H2O2. Taken together, these results suggest that most oral streptococci except S. mutans efficiently induce damage to human PDL cells by inducing apoptotic cell death with bacterial H2O2 and lipoproteins, which might contribute to the progression of oral infectious diseases such as apical periodontitis.
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Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.
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Imunidade Celular , Imunidade Humoral , Doenças das Aves Domésticas/prevenção & controle , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/imunologia , Vacinas de Produtos Inativados/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Citocinas/metabolismo , Imunização , Lipopolissacarídeos/imunologia , Camundongos , Vacinas contra Salmonella/administração & dosagem , Salmonella enterica/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos da radiaçãoRESUMO
Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to ß-lactams and for those who are pregnant.
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Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Genes MDR/genética , Genótipo , Família Multigênica , Streptococcus agalactiae/genética , Células A549 , Animais , Proteínas de Bactérias/genética , Genoma Bacteriano , Humanos , Masculino , Meningites Bacterianas/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/classificação , VirulênciaRESUMO
Expression of bacteriophage lysinSM1 by Streptococcus oralis strain SF100 is thought to be important for the pathogenesis of infective endocarditis, due to its ability to mediate bacterial binding to fibrinogen. To better define the lysinSM1 binding site on fibrinogen Aα, and to investigate the impact of binding on fibrinolysis, we examined the interaction of lysinSM1 with a series of recombinant fibrinogen Aα variants. These studies revealed that lysinSM1 binds the C-terminal region of fibrinogen Aα spanned by amino acid residues 534 to 610, with an affinity of equilibrium dissociation constant (KD) of 3.23 × 10-5 M. This binding site overlaps the known binding site for plasminogen, an inactive precursor of plasmin, which is a key protease responsible for degrading fibrin polymers. When tested in vitro, lysinSM1 competitively inhibited plasminogen binding to the αC region of fibrinogen Aα. It also inhibited plasminogen-mediated fibrinolysis, as measured by thromboelastography (TEG). These results indicate that lysinSM1 is a bi-functional virulence factor for streptococci, serving as both an adhesin and a plasminogen inhibitor. Thus, lysinSM1 may facilitate the attachment of bacteria to fibrinogen on the surface of damaged cardiac valves and may also inhibit plasminogen-mediated lysis of infected thrombi (vegetations) on valve surfaces. IMPORTANCE The interaction of streptococci with human fibrinogen and platelets on damaged endocardium is a central event in the pathogenesis of infective endocarditis. Streptococcus oralis can bind platelets via the interaction of bacteriophage lysinSM1 with fibrinogen on the platelet surface, and this process has been associated with increased virulence in an animal model of endocarditis. We now report that lysinSM1 binds to the αC region of the human fibrinogen Aα chain. This interaction blocks plasminogen binding to fibrinogen and inhibits fibrinolysis. In vivo, this inhibition could prevent the lysis of infected vegetations, thereby promoting bacterial persistence and virulence.
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Fibrinogênio/metabolismo , Fibrinólise , Plasminogênio/metabolismo , Fagos de Streptococcus/fisiologia , Streptococcus/metabolismo , Sítios de Ligação , Endocardite/microbiologia , Fibrina/química , Fibrina/metabolismo , Humanos , Ligação Proteica , Streptococcus/genética , Streptococcus/patogenicidade , Streptococcus/virologia , Fagos de Streptococcus/genética , VirulênciaRESUMO
Streptococcus pneumoniae (pneumococcus) can cause respiratory and systemic diseases. Recently, γ-irradiation-inactivated, non-encapsulated, intranasal S. pneumoniae (r-SP) vaccine has been introduced as a novel serotype-independent and cost-effective vaccine. However, the immunogenic mechanism of r-SP is poorly understood. Here, we comparatively investigated the protective immunity and immunogenicity of r-SP to the heat-(h-SP) or formalin-inactivated vaccine (f-SP) without adjuvants. Mice were intranasally immunized with each vaccine three times and then challenged with a lethal dose of S. pneumoniae TIGR4 strain and then subsequently evaluated for their immune responses. Immunization with r-SP elicited modestly higher protection against S. pneumoniae than h-SP or f-SP. Immunization with r-SP enhanced pneumococcal-specific IgA in the nasal wash and IgG in bronchoalveolar lavage fluid. Immunization with r-SP enhanced S. pneumoniae-specific IgG, IgG1, and IgG2b in the serum. r-SP more potently induced the maturation of dendritic cells in the cervical lymph nodes than h-SP or f-SP. Interestingly, populations of follicular helper T cells and IL-4-producing cells were potently increased in cervical lymph nodes of r-SP-immunized mice. Collectively, r-SP could be an effective intranasal, inactivated whole-cell vaccine in that it elicits S. pneumoniae-specific antibody production and follicular helper T cell activation leading to protective immune responses against S. pneumoniae infection.
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The most widely used influenza vaccines are prepared by chemical inactivation. However, chemical, especially formalin, treatment-induced modifications of the antigenic structure of the virus are frequently associated with adverse effects including low efficacy of protection, unexpected immune responses, or exacerbation of disease. Gamma-irradiation was suggested as an alternative influenza virus inactivation method due to its great features of completely inactivating virus while not damaging the structures of protein antigens, and cross-protective ability against heterologous strains. However, immunological features of gamma radiation-inactivated influenza vaccine have not been fully understood. In this study, we aimed to investigate the humoral and cellular immune responses of gamma radiation-inactivated influenza vaccine. The gamma irradiation-inactivated influenza vaccine (RADVAXFluA) showed complete viral inactivation but retained normal viral structure with functional activities of viral protein antigens. Intranasal immunization of RADVAXFluA provided better protection against influenza virus infection than formalin-inactivated influenza virus (FIV) in mice. RADVAXFluA greatly enhanced the production of virus-specific serum IgG and alveolar mucosal IgA, which effectively neutralized HA (hemagglutinin) and NA (neuraminidase) activities, and blocked viral binding to the cells, respectively. Further analysis of IgG subclasses showed RADVAXFluA-immunized sera had higher levels of IgG1 and IgG2a than those of FIV-immunized sera. In addition, analysis of cellular immunity found RADVAXFluA induced strong dendritic cells (DC) activation resulting in higher DC-mediated activation of CD8+ T cells than FIV. The results support improved immunogenicity by RADVAXFluA.
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Vacinas contra Influenza , Infecções por Orthomyxoviridae , Administração Intranasal , Animais , Anticorpos Antivirais , Linfócitos T CD8-Positivos , Raios gama , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Produtos InativadosRESUMO
Deinococcus radiodurans is an extremely resistant bacterium against extracellular stress owing to on its unique physiological functions and the structure of its cellular constituents. Interestingly, it has been reported that the pattern of alteration in Deinococcus proportion on the skin is negatively correlated with skin inflammatory diseases, whereas the proportion of Staphylococcus aureus was increased in patients with chronic skin inflammatory diseases. However, the biological mechanisms of deinococcal interactions with other skin commensal bacteria have not been studied. In this study, we hypothesized that deinococcal cellular constituents play a pivotal role in preventing S. aureus colonization by inhibiting biofilm formation. To prove this, we first isolated cellular constituents, such as exopolysaccharide (DeinoPol), cell wall (DeinoWall), and cell membrane (DeinoMem), from D. radiodurans and investigated their inhibitory effects on S. aureus colonization and biofilm formation in vitro and in vivo. Among them, only DeinoPol exhibited an anti-biofilm effect without affecting bacterial growth and inhibiting staphylococcal colonization and inflammation in a mouse skin infection model. Moreover, the inhibitory effect was impaired in the Δdra0033 strain, a mutant that cannot produce DeinoPol. Remarkably, DeinoPol not only interfered with S. aureus biofilm formation at early and late stages but also disrupted a preexisting biofilm by inhibiting the production of poly-N-acetylglucosamine (PNAG), a key molecule required for S. aureus biofilm formation. Taken together, the present study suggests that DeinoPol is a key molecule in the negative regulation of S. aureus biofilm formation by D. radiodurans. Therefore, DeinoPol could be applied to prevent and/or treat infections or inflammatory diseases associated with S. aureus biofilms.
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Salmonella enterica serovar Typhimurium causes invasive nontyphoidal Salmonella diseases in animals and humans, resulting in a high mortality rate and huge economic losses globally. As the prevalence of antibioticresistant Salmonella has been increasing, vaccination is thought to be the most effective and economical strategy to manage salmonellosis. The present study aimed to investigate whether dysfunction in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), which is critical for carbon uptake and survival in macrophages, may be adequate to generate Salmonellaattenuated vaccine strains. A Salmonella strain (KST0555) was generated by deleting the ptsI gene from the PTS and it was revealed that this auxotrophic mutant was unable to efficiently utilize predominant carbon sources during infection (glucose and glycerol), reduced its invasion and replication capacity in macrophages, and significantly (P=0.0065) lowered its virulence in the setting of a mouse colitis model, along with a substantially decreased intestinal colonization and invasiveness compared with its parent strain. The reverse transcriptionquantitative PCR results demonstrated that the virulence genes in Salmonella pathogenicity island-1 (SPI-1) and -2 (SPI-2) and the motility of KST0555 were all downregulated compared with its parent strain. Finally, it was revealed that when mice were immunized orally with live KST0555, Salmonellaspecific humoral and cellular immune responses were effectively elicited, providing protection against Salmonella infection. Thus, the present promising data provides a strong rationale for the advancement of KST0555 as a live Salmonella vaccine candidate and ptsI as a potential target for developing a live attenuated bacterial vaccine strain.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Fosfotransferases/genética , Fosfotransferases/imunologia , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Vacinas Atenuadas/imunologia , Animais , Colite/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunização/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Virulência/imunologiaRESUMO
Streptococcus pneumoniae is the most common respiratory bacterial pathogen among cases of community-acquired infection in young children, older adults, and individuals with underlying medical conditions. Although capsular polysaccharide-based pneumococcal vaccines have contributed to significant decrease in invasive pneumococcal infections, these vaccines have some limitations, including limited serotype coverage, lack of effective mucosal antibody responses, and high costs. In this study, we investigated the safety and immunogenicity of a live, whole-cell pneumococcal vaccine constructed by deleting the gene for prolipoprotein diacylglyceryl transferase (lgt) from the encapsulated pneumococcal strain TIGR4 (TIGR4Δlgt) for protection against heterologous pneumococcal strains. Pneumococcal strain TIGR4 was successfully attenuated by deletion of lgt, resulting in the loss of inflammatory activity and virulence. TIGR4Δlgt colonized the nasopharynx long enough to induce strong mucosal IgA and IgG2b-dominant systemic antibody responses that were cross-reactive to heterologous pneumococcal serotypes. Finally, intranasal immunization with TIGR4Δlgt provided serotype-independent protection against pneumococcal challenge in mice. Taken together, our results suggest that TIGR4Δlgt is an avirulent and attractive broad-spectrum pneumococcal vaccine candidate. More broadly, we assert that modulation of such "master" metabolic genes represents an emerging strategy for developing more effective vaccines against numerous infectious agents.
Assuntos
Proteínas de Bactérias/imunologia , Imunogenicidade da Vacina , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Transferases/deficiência , Administração Intranasal , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/imunologia , Proteínas de Bactérias/genética , Genes Bacterianos , Imunidade nas Mucosas , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nasofaringe/imunologia , Nasofaringe/microbiologia , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/administração & dosagem , Células RAW 264.7 , Sorogrupo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Transferases/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologia , VirulênciaRESUMO
INTRODUCTION: Apical periodontitis is an inflammatory disease in the periradicular region of teeth that results from infection by multispecies bacterial biofilm residing in the root canal system. In this study, we investigated whether Lactobacillus plantarum lipoteichoic acid (Lp.LTA) could inhibit multispecies oral pathogenic bacterial biofilm formation. METHODS: Highly pure and structurally intact Lp.LTA was purified from L. plantarum. Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were co-cultured to form oral multispecies biofilm in the presence or absence of Lp.LTA on culture plates or human dentin slices. Preformed biofilm was treated with or without Lp.LTA, followed by additional treatment with intracanal medicaments such as calcium hydroxide or chlorhexidine digluconate. Confocal microscopy and crystal violet assay were performed to determine biofilm formation. Biofilm on human dentin slices was visualized with a scanning electron microscope. RESULTS: Biofilm formation of multispecies bacteria on the culture dishes was dose-dependently reduced by Lp.LTA compared with the nontreatment control group. Lp.LTA also inhibited multispecies biofilm formation on the dentin slices in a dose-dependent manner. Interestingly, Lp.LTA was shown to reduce preformed multispecies biofilm compared with the nontreatment group. Moreover, Lp.LTA potentiated the effectiveness of the intracanal medicaments in the removal of preformed multispecies biofilm. CONCLUSIONS: These results suggest that Lp.LTA is a potential anti-biofilm agent for treatment or prevention of oral infectious disease, including apical periodontitis, which is mainly caused by multispecies bacterial biofilm.
Assuntos
Actinomyces/fisiologia , Biofilmes/efeitos dos fármacos , Enterococcus faecalis/fisiologia , Lactobacillus plantarum/química , Ligilactobacillus salivarius/fisiologia , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/farmacologia , Ácidos Teicoicos/isolamento & purificação , Ácidos Teicoicos/farmacologia , Actinomyces/patogenicidade , Hidróxido de Cálcio/farmacologia , Clorexidina/análogos & derivados , Clorexidina/farmacologia , Dentina/microbiologia , Depressão Química , Relação Dose-Resposta a Droga , Enterococcus faecalis/patogenicidade , Humanos , Ligilactobacillus salivarius/patogenicidade , Lipopolissacarídeos/uso terapêutico , Periodontite Periapical/tratamento farmacológico , Periodontite Periapical/microbiologia , Periodontite Periapical/prevenção & controle , Irrigantes do Canal Radicular/farmacologia , Ácidos Teicoicos/uso terapêuticoRESUMO
Enterococcus faecalis, a Gram-positive bacterium commonly isolated in patients with refractory apical periodontitis, invades dentin tubules easily and forms biofilms. Bacteria in biofilms, which contribute to recurrent and/or chronic inflammatory diseases, are more resistant to antimicrobial agents than planktonic cells and easily avoid phagocytosis. Although Lactobacillus plantarum lipoteichoic acid (Lp.LTA) is associated with biofilm formation, the effect of Lp.LTA on biofilm formation by E. faecalis is not clearly understood. In this study, we investigated whether Lp.LTA inhibits E. faecalis biofilm formation. The degree of biofilm formation was determined by using crystal violet assay and LIVE/DEAD bacteria staining. The quantification of bacterial growth was determined by measuring the optical density at 600 nm with a spectrophotometer. Formation of biofilms on human dentin slices was observed under a scanning electron microscope. E. faecalis biofilm formation was reduced by Lp.LTA treatment in a dose-dependent manner. Lp.LTA inhibited biofilm development of E. faecalis at the early stage without affecting bacterial growth. LTA from other Lactobacillus species such as Lactobacillus acidophilus, Lactobacillus casei, or Lactobacillus rhamnosus GG also inhibited E. faecalis biofilm formation. In particular, among LTAs from various lactobacilli, Lp.LTA showed the highest inhibitory effect on biofilms formed by E. faecalis. Interestingly, LTAs from lactobacilli could remove the biofilm preformed by E. faecalis. These inhibitory effects were also observed on the surface of human dentin slices. In conclusion, Lactobacillus species LTA inhibits biofilm formation caused by E. faecalis and it could be used as an anti-biofilm agent for prevention or treatment against E. faecalis-associated diseases.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Lactobacillus/química , Lipopolissacarídeos/farmacologia , Ácidos Teicoicos/farmacologia , Antibacterianos/metabolismo , Dentina/microbiologia , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Lactobacillus/metabolismo , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Ácidos Teicoicos/metabolismo , Doenças Dentárias/microbiologiaRESUMO
Salmonella enterica is a major human pathogen that causes invasive non-typhoidal Salmonellosis (iNTS), resulting in significant morbidity and mortality. Although a number of pre-clinical and clinical studies have reported on the feasibility of developing a safe and effective vaccine against iNTS, there have been no licensed Salmonella vaccines available to protect against NTS strains. Vaccine formulations of highest priority for NTS are live attenuated vaccines, which can elicit effective induction of intestinal mucosal and intracellular bacteria-specific cell mediated immune responses. Since glucose is crucial for intracellular survival and replication in host cells, we constructed strains with mutations in components of the glucose uptake system, called the phosphotransferase system (PTS), and compared the relative virulence and immune responses in mice. In this study, we found that the strain with mutations in both ptsI and crr (KST0556) was the most attenuated strain among the tested strains, and proved to be highly effective in inducing a mucosal immune response that can protect against NTS infections in mice. Thus, we suggest here that KST0556 (ΔptsIΔcrr) is a potential live vaccine candidate for NTS, and may also be a candidate for a live delivery vector for heterologous antigens. Moreover, since PTS is a well-conserved glucose transporter system in both Gramnegative and Gram-positive bacteria, the ptsI and crr genes may be potential targets for creating live bacterial vectors or vaccine strains.
Assuntos
Proteínas de Bactérias/imunologia , Desoxirribonucleases de Sítio Específico do Tipo II/imunologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/imunologia , Infecções por Salmonella/prevenção & controle , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/enzimologia , Vacinas Atenuadas/imunologia , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Desoxirribonucleases de Sítio Específico do Tipo II/administração & dosagem , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Deleção de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/administração & dosagem , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Vacinas contra Salmonella/administração & dosagem , Vacinas contra Salmonella/genética , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genéticaRESUMO
Gram-positive bacteria such as Streptococcus gordonii causing life-threatening infective endocarditis are mainly recognized by Toll-like receptor 2 (TLR2). Lipoteichoic acid (LTA) and lipoproteins are representative TLR2 ligands that play important roles in bacterial infection and in host inflammatory responses. In the present study, we generated an LTA-deficient mutant (ΔltaS) and a lipoprotein-deficient mutant (Δlgt) and investigated the contributions of LTA and lipoproteins to bacterial morphology and their effect on induction of proinflammatory cytokines in THP-1 and mouse bone-marrow derived macrophages (BMDMs). Deletion of ltaS and lgt was confirmed by PCR analysis of genomic DNA from each mutant. The mutants with absence of LTA or lipoproteins were examined by SDS-PAGE followed by Western blotting with anti-LTA antibodies and silver staining, respectively. Interestingly, scanning and transmission electron microscopies showed no difference in the bacterial cell morphology or size between the wild-type and the mutants even though substantial changes in the cell size and/or morphology have been reported in other Gram-positive bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis. However, S. gordonii wild-type and ΔltaS potently induced the expression of proinflammatory cytokines including TNF-α, IL-8, and IL-1ß at the mRNA and protein levels, while Δlgt did not have these effects. Furthermore, lipoproteins purified from S. gordonii also induced the expression of the aforementioned cytokines more potently than the purified LTA. Neither LTA nor lipoprotein induced TNF-α, KC (IL-8 counterpart in mouse), and IL-1ß in TLR2-deficient BMDMs. S. gordonii Δlgt was less virulent than the wild-type or ΔltaS in a mouse intraperitoneal infection model. Collectively, these results suggest that S. gordonii lipoproteins, but not LTA, are mainly responsible for the infection and inflammatory responses.