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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638530

RESUMO

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Vesículas Extracelulares/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Vacinas Bacterianas/biossíntese , Feminino , Lipopolissacarídeos/imunologia , Camundongos , Neisseria meningitidis/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Vacinas Protozoárias/biossíntese , Salmonella typhimurium/imunologia , Shigella sonnei/imunologia
2.
FASEB J ; 35(11): e21942, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34670018

RESUMO

Atherosclerosis is a chronic inflammatory disease. Pathophysiological similarities between chronic infections and atherosclerosis triggered interests between these conditions. The seroepidemiological study showed that Helicobacter pylori strains that express cytotoxin-associated gene A (CagA), an oncoprotein and a major virulence factor, was positively correlated with atherosclerosis and related clinical events. Nevertheless, the underlying mechanism is poorly understood. In this study, the seroprevalence of infection by H. pylori and by strains express CagA assessed by enzyme-linked immunosorbent assay (ELISA) showed that the prevalence of CagA strains rather than H. pylori in patients was positively correlated with atherogenesis. Correspondingly, we found that CagA augmented the growth of plaque of ApoE-/- mice in the early stage of atherosclerosis and promoted the expression of adhesion molecules and inflammatory cytokines in mouse aortic endothelial cells (MAECs). Mechanistically, both si-NLRP3 and si-IL-1ß mitigated the promoting effect of CagA on the inflammatory activation of HAECs. In vivo, the inhibition of NLRP3 by MCC950 significantly attenuated the promoting effect of CagA on plaque growth of ApoE-/- mice. We also propose NLRP3 as a potential therapeutic target for CagA-positive H. pylori infection-related atherosclerosis and emphasize the importance of inflammation in atherosclerosis pathology.


Assuntos
Antígenos de Bactérias/metabolismo , Aorta/patologia , Aterosclerose/sangue , Proteínas de Bactérias/metabolismo , Caspase 1/metabolismo , Células Endoteliais/metabolismo , Infecções por Helicobacter/sangue , Helicobacter pylori/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Placa Aterosclerótica/sangue , Idoso , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Aorta/metabolismo , Aterosclerose/microbiologia , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Feminino , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Placa Aterosclerótica/microbiologia , Estudos Soroepidemiológicos , Células THP-1
3.
Front Immunol ; 12: 726920, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34671347

RESUMO

Background: The lack of suitable diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. Serological tests, based on multiple target antigens, represent an attractive option for diagnosis of this disease due to their rapidity, convenience, and low cost. Methods: Measures to reduce non-specific reactions and thereby improve the specificity of serological tests were investigated, including blocking antibodies against common bacteria in serum samples and synthesizing polypeptides covering non-conserved dominant B-cell epitopes of antigens. In addition, a fusion polyprotein containing HspX and eight other antigen sequences was constructed and expressed to increase overall sensitivity of the tests. Results: Inclusion of Escherichia coli lysate partially increased the specificity of the serological tests, while synthesis and inclusion of peptides containing non-conserved sequences of TB antigens as well as dominant B-cell epitopes reduced non-specific reactions without a decrease in sensitivity of the tests. A polyprotein fusing HspX and eight other antigen sequences was constructed and displayed 60.2% sensitivity, which was higher than that of HspX and the other individual antigen segments. Moreover, the specificity of the polyprotein was 93.8%, which was not significantly decreased when compared with HspX and the other individual antigen segments. Conclusions: The roles of the fusion polyprotein in the humoral immune response against TB infection were demonstrated and provide a potential novel approach for the development of TB diagnostics.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Poliproteínas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Tuberculose/diagnóstico , Adsorção , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Bactérias/química , Bactérias/genética , Bactérias/imunologia , Proteínas de Bactérias/genética , Sequência de Bases , Epitopos de Linfócito B/imunologia , Humanos , Imunidade Humoral , Imunoglobulina G/imunologia , Poliproteínas/genética , Testes Sorológicos , Tuberculose/imunologia
4.
J Immunol Methods ; 499: 113168, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34673004

RESUMO

Various Leptospira components have been identified as candidate antigens for the detection of antibody to Leptospira. LipL32 is a Leptospira membrane protein which has been widely studied. The report of Leptospira whole-genome sequencing demonstrated that pathogenic Leptospira contained the nucleotide sequence (colA gene) coding for the collagenase. Expression of ColA protein and its enzymatic activity was demonstrated. In this study, cloned ColA protein, in comparison with LipL32, was used as an antigen for antibody detection. Thirty pairs of sera from human leptospirosis patients were tested. Sera from blood donors, and patients with scrub typhus and dengue virus infection (20 samples from each group) were tested for the specificity. All sera from leptospirosis patients tested in this study reacted with both ColA and LipL32 proteins. Sera from blood donors, patients with scrub typhus and dengue virus infection did not react with ColA protein. Data suggested that sensitivity and specificity of ColA protein for Leptospira antibody detection were 100%. In addition, ColA protein showed higher specificity than LipL32. Our data suggested that ColA protein could be another candidate antigen for antibody detection in leptospirosis diagnosis.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Colagenases/metabolismo , Testes Imunológicos , Leptospira/enzimologia , Leptospirose/diagnóstico , Lipoproteínas/imunologia , Animais , Cricetinae , Humanos , Leptospirose/imunologia
5.
PLoS One ; 16(10): e0258317, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34634075

RESUMO

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.


Assuntos
Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Bacillus anthracis/imunologia , Cápsulas Bacterianas/imunologia , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Choque Térmico/imunologia , Animais , Vacinas contra Antraz/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Choque Térmico/isolamento & purificação , Cavalos , Imunoglobulina G/imunologia , Plasmídeos/metabolismo , Homologia de Sequência de Aminoácidos , Esporos Bacterianos/imunologia
6.
PLoS One ; 16(10): e0257920, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34618810

RESUMO

Tuberculosis (TB), a contagious disease mainly caused by Mycobacterium tuberculosis (M. tb), Mycobacterium bovis (M. bovis), and Mycobacterium caprae (M. caprae), poses a major global threat to the health of humans and many species of animals. Developing an ante-mortem detection technique for different species would be of significance in improving the surveillance employing a One Health strategy. To achieve this goal, a universal indirect ELISA was established for serologically detecting Mycobacterium tuberculosis complex infection for multiple live hosts by using a fusion protein of MPB70, MPB83, ESAT6, and CFP10 common in M. tb, M. bovis, and M. caprae as the coating antigen (MMEC) and HRP-labeled fusion protein A and G as a secondary antibody. After testing the known positive and negative sera, the receiver operating characteristic curves were constructed to decide the cut-off values. Then, the diagnostic sensitivity and specificity of MMEC/AG-iELISA were determined as 100.00% (95% CI: 96.90%, 100.00%) and 100.00% (95% CI: 98.44%, 100.00%) for M. bovis infection of cattle, 100.00% (95% CI: 95.00%, 100.00%) and 100.0% (95% CI: 96.80%, 100.00%) for M. bovis infection of sheep, 90.74% (95% CI: 80.09%, 95.98%) and 98.63% (95% CI: 95.14%, 99.76%) for M. bovis infection of cervids, 100.00% (95% CI: 15.81%, 100.00%) and 98.81% (95% CI: 93.54%, 99.97%) for M. bovis infection of monkeys, 100.00% (95% CI: 86.82%, 100.00%) and 94.85% (95% CI: 91.22%, 97.03%) for M. tb infection of humans. Furthermore, this MMEC/AG-iELISA likely detects M. caprae infection in roe deer. Thus this method has a promising application in serological TB surveillance for multiple animal species thereby providing evidence for taking further action in TB control.


Assuntos
Ensaio de Imunoadsorção Enzimática , Mycobacterium tuberculosis/isolamento & purificação , Testes Sorológicos , Tuberculose/diagnóstico , Animais , Animais Selvagens/microbiologia , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Bovinos , Cervos/microbiologia , Testes Diagnósticos de Rotina , Humanos , Mycobacterium bovis/isolamento & purificação , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/genética , Ovinos/microbiologia , Tuberculose/microbiologia
7.
MAbs ; 13(1): 1978130, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34586015

RESUMO

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Microfluídica/métodos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Preservação de Sangue , COVID-19/imunologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Hibridomas/imunologia , Separação Imunomagnética , Dispositivos Lab-On-A-Chip , Camundongos , Microfluídica/instrumentação , Muromonab-CD3/imunologia , Plasmócitos , Proteínas Recombinantes/imunologia , SARS-CoV-2/imunologia , Toxoide Tetânico/imunologia , Vacinação
8.
PLoS One ; 16(9): e0253514, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34499659

RESUMO

Multiple different recombinant and peptide antigens are now available for serodiagnosis of Lyme disease (LD), but optimizing test utilization remains challenging. Since 1995 the Centers for Disease Control and Prevention (CDC) has recommended a 2-tiered serologic approach consisting of a first-tier whole-cell enzyme immunoassay (EIA) for polyvalent antibodies to Borrelia burgdorferi followed by confirmation of positive or equivocal results by IgG and IgM immunoblots [standard 2-tiered (STT) approach]. Newer modified 2-tiered (MTT) approaches employ a second-tier EIA to detect antibodies to B. burgdorferi rather than immunoblotting. We applied modern bioinformatic techniques to a large public database of recombinant and peptide antigen-based immunoassays to improve testing strategy. A retrospective CDC collection of 280 LD samples and 559 controls had been tested using the STT approach as well as kinetic-EIAs for VlsE1-IgG, C6-IgG, VlsE1-IgM, and pepC10-IgM antibodies. When used individually, the cutoff for each kinetic-EIA was set to generate 99% specificity. Utilizing logistic-likelihood regression analysis and receiver operating characteristic (ROC) techniques we determined that VlsE1-IgG, C6-IgG, and pepC10-IgM antibodies each contributed significant diagnostic information; a single-tier diagnostic score (DS) was generated for each sample using a weighted linear combination of antibody levels to these 3 antigens. DS performance was then compared to the STT and to MTT models employing different combinations of kinetic-EIAs. After setting the DS cutoff to match STT specificity (99%), the DS was 22.5% more sensitive than the STT for early-acute-phase disease (95% CI: 11.8% to 32.2%), 16.0% more sensitive for early-convalescent-phase disease (95% CI: 7.2% to 24.7%), and equivalent for detection of disseminated infection. The DS was also significantly more sensitive for early-acute-phase LD than MTT models whose specificity met or exceeded 99%. Prospective validation of this single-tier diagnostic score for Lyme disease will require larger studies using a broader range of potential cross-reacting conditions.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/metabolismo , Borrelia burgdorferi/imunologia , Biologia Computacional/métodos , Doença de Lyme/diagnóstico , Estudos de Casos e Controles , Testes Diagnósticos de Rotina , Diagnóstico Precoce , Humanos , Modelos Logísticos , Doença de Lyme/imunologia , Estudos Retrospectivos , Sensibilidade e Especificidade
9.
Front Immunol ; 12: 685742, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34512624

RESUMO

Background: Pregnancy is a portentous stage in life, during which countless events are precisely orchestrated to ensure a healthy offspring. Maternal microbial communities are thought to have a profound impact on development. Although antibiotic drugs may interfere in these processes, they constitute the most frequently prescribed medication during pregnancy to prohibit detrimental consequences of infections. Gestational antibiotic intervention is linked to preeclampsia and negative effects on neonatal immunity. Even though perturbations in the immune system of the mother can affect reproductive health, the impact of microbial manipulation on maternal immunity is still unknown. Aim: To assess whether antibiotic treatment influences maternal immunity during pregnancy. Methods: Pregnant mice were treated with broad-spectrum antibiotics. The maternal gut microbiome was assessed. Numerous immune parameters throughout the maternal body, including placenta and amniotic fluid were investigated and a novel machine-learning ensemble strategy was used to identify immunological parameters that allow distinction between the control and antibiotic-treated group. Results: Antibiotic treatment reduced diversity of maternal microbiota, but litter sizes remained unaffected. Effects of antibiotic treatment on immunity reached as far as the placenta. Four immunological features were identified by recursive feature selection to contribute to the most robust classification (splenic T helper 17 cells and CD5+ B cells, CD4+ T cells in mesenteric lymph nodes and RORγT mRNA expression in placenta). Conclusion: In the present study, antibiotic treatment was able to affect the carefully coordinated immunity during pregnancy. These findings highlight the importance of inclusion of immunological parameters when studying the effects of medication used during gestation.


Assuntos
Imunidade Adaptativa/imunologia , Animais Recém-Nascidos/imunologia , Anticorpos Antibacterianos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Microbioma Gastrointestinal/imunologia , Animais , Animais Recém-Nascidos/microbiologia , Antibacterianos/farmacologia , Feminino , Microbioma Gastrointestinal/genética , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Intestinos/microbiologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Gravidez
10.
J Immunol ; 207(4): 1138-1149, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34341168

RESUMO

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. One obstacle to the development of M protein-based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Reações Cruzadas/imunologia , Vacinas Estreptocócicas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Peptídeos/imunologia , Vacinas Sintéticas/imunologia
11.
Biomed Environ Sci ; 34(7): 528-539, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34353416

RESUMO

Objectives: To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M. intracellulare and M. tuberculosis. Methods: Protein extracts from M. intracellulare were used to immunize BALB/c mice. The antigens were evaluated using cellular and humoral immunoassays. The common genes between M. intracellular and M. tuberculosis were identified using genome-wide comparative analysis, and cross-reactive proteins were screened using immunoproteome microarrays. Results: Immunization with M. intracellulare proteins induced significantly higher levels of the cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-6 (IL-6) and immunoglobulins IgG, IgG1, IgM, and IgG2a in mouse serum. Bone marrow-derived macrophages isolated from mice immunized with M. intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants. Whole-genome sequence analysis revealed 396 common genes between M. intracellulare and M. tuberculosis. Microchip hybridization with M. tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M. intracellulare protein extracts. Sixty common antigens were found using both microchip and genomic comparative analyses. Conclusion: This is the advanced study to investigate the immunogenicity of M. intracellulare proteins and the cross-reactive proteins between M. intracellulare and M. tuberculosis. The results revealed the presence of a number of cross-reactive proteins between M. intracellulare and M. tuberculosis. Therefore, this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M. intracellulare and M. tuberculosis in future.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Complexo Mycobacterium avium/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Reações Cruzadas , Citocinas/imunologia , Feminino , Genoma Bacteriano , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Macrófagos/imunologia , Camundongos Endogâmicos BALB C , Complexo Mycobacterium avium/genética , Mycobacterium tuberculosis/genética , Vacinas contra a Tuberculose/administração & dosagem , Sequenciamento Completo do Genoma
12.
Ann Clin Lab Sci ; 51(4): 540-545, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34452893

RESUMO

OBJECTIVE: Mycoplasma pneumoniae (M. pneumoniae), an extracellular pathogen lacking a cell wall, causes respiratory infection in adults and children and has been implicated in asthma exacerbation; immunoglobulin (Ig) E may be involved in these exacerbations. Specific IgM and IgG immune response to M. pneumoniae has been reported, but less is known about IgE M. pneumoniae antibody (Ab) responses in asthma. Previous studies in our laboratory demonstrated that asthmatic children have increased IgM M. pneumoniae levels, but not IgE. Thus, we sought to investigate whether past M. pneumoniae infection triggers production of M. pneumoniae-specific IgE Abs in adult subjects with/without asthma. METHODS: M. pneumoniae- IgE and -IgM Ab responses were studied in adult asthmatic (N=22) and non-asthmatic (N=22) subjects (ELISA). Data are reported as antibody index. Threshold detection levels: IgE, IgM: 0.2, 0.9, respectively. RESULTS: M. pneumoniae-IgE Ab levels were low and below the threshold of detection in both asthmatic and non-asthmatics (0.002±0.008 vs. 0.02±0.03; P=0.021). However, specific-IgM levels were slightly higher in non-asthmatics compared with asthmatics (0.96±0.37 vs. 0.79±0.31; P=0.054). M. pneumoniae-IgM Ab positivity was similar in both groups (P=1.0). CONCLUSION: IgM M. pneumoniae Abs may play an important role in non-asthma and persist for months after acute infection. IgE M. pneumoniae Abs may play a less important role in both groups.


Assuntos
Anticorpos Antibacterianos/sangue , Asma/imunologia , Imunoglobulina E/imunologia , Imunoglobulina M/sangue , Mycoplasma pneumoniae/imunologia , Pneumonia por Mycoplasma/imunologia , Adulto , Anticorpos Antibacterianos/imunologia , Asma/sangue , Asma/complicações , Asma/epidemiologia , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , New York/epidemiologia , Pneumonia por Mycoplasma/sangue , Pneumonia por Mycoplasma/epidemiologia , Pneumonia por Mycoplasma/etiologia , Prognóstico
13.
Sci Rep ; 11(1): 16672, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404881

RESUMO

Immune response involving various immunoglobulin (Ig) isotypes and subtypes to microbiome is involved in the pathogenesis and disease activity of inflammatory bowel diseases (IBDs). To clarify the presence of Ig-coated bacteria in the intestine and its association with disease activity in ulcerative colitis (UC) and Crohn's disease (CD), we extracted and classified Ig-coated bacteria from fecal samples of 42 patients with IBD and 12 healthy controls (HCs) using flow cytometry and 16S ribosomal RNA sequence analysis. The percentage of bacteria coated with IgA and IgM was higher in patients with IBD than in HCs, and IgG-coated bacteria were found only in patients with IBD. Moreover, the percentages of bacteria coated with IgG1, IgG2, IgG3, and IgM in UC samples and IgG3, IgG4, and IgM in CD samples were correlated with disease activities. The proportions of Bacteroides ovatus and Streptococcus increased during the active phase of CD. Hence, the detailed analysis of Ig-coated bacteria and Ig subtypes using flow cytometry could aid in developing useful indicators of disease activity and identifying more disease-related bacteria, which could become novel treatment targets for IBDs.


Assuntos
Bactérias/imunologia , Isotipos de Imunoglobulinas/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Adulto , Anticorpos Antibacterianos/imunologia , Fezes/microbiologia , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Doenças Inflamatórias Intestinais/imunologia , Masculino , Pessoa de Meia-Idade
14.
PLoS Pathog ; 17(8): e1009905, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34449811

RESUMO

B1 cells, a subset of B lymphocytes whose developmental origin, phenotype, and function differ from that of conventional B2 cells, are the main source of "natural" IgM but can also respond to infection by rapidly producing pathogen-specific IgM directed against T-independent antigens. Francisella tularensis (Ft) is a Gram-negative bacterium that causes tularemia. Infection with Ft Live Vaccine Strain activates B1 cells for production of IgM directed against the bacterial LPS in a process incompletely understood. Here we show that immunization with purified Ft LPS elicits production of LPS-specific IgM and IgG3 by B1 cells independently of TLR2 or MyD88. Immunization, but not infection, generated peritoneum-resident memory B1 cells that differentiated into LPS-specific antibody secreting cells (ASC) upon secondary challenge. IL-5 was rapidly induced by immunization with Ft LPS and was required for production of LPS-specific IgM. Antibody-mediated depletion of ILC2 indicated that these cells were the source of IL-5 and were required for IgM production. IL-25, an alarmin that strongly activates ILC2, was rapidly secreted in response to immunization or infection and its administration to mice significantly increased IgM production and B1 cell differentiation to ASC. Conversely, mice lacking IL-17RB, the IL-25 receptor, showed impaired IL-5 induction, IgM production, and B1 ASC differentiation in response to immunization. Administration of IL-5 to Il17rb-/- mice rescued these B1 cells-mediated responses. Il17rb-/- mice were more susceptible to infection with Ft LVS and failed to develop immunity upon secondary challenge suggesting that LPS-specific IgM is one of the protective adaptive immune mechanisms against tularemia. Our results indicated that immunization with Ft LPS triggers production of IL-25 that, through stimulation of IL-5 release by ILC2, promotes B1 cells activation and differentiation into IgM secreting cells. By revealing the existence of an IL-25-ILC2-IL-5 axis our results suggest novel strategies to improve vaccination against T-independent bacterial antigens.


Assuntos
Anticorpos Antibacterianos/imunologia , Subpopulações de Linfócitos B/imunologia , Francisella tularensis/imunologia , Imunoglobulina M/imunologia , Interleucina-5/metabolismo , Interleucinas/metabolismo , Lipopolissacarídeos/farmacologia , Animais , Anticorpos Antibacterianos/metabolismo , Subpopulações de Linfócitos B/metabolismo , Imunidade Inata , Imunoglobulina M/metabolismo , Interleucina-5/genética , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/fisiologia , Receptores de Interleucina-17/fisiologia , Receptor 2 Toll-Like/fisiologia , Tularemia/imunologia , Tularemia/microbiologia , Tularemia/patologia
15.
Mol Immunol ; 138: 48-57, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34343723

RESUMO

Knowledge of immunodominant B-cell epitopes is essential to design powerful diagnostic strategies aiming for antibody detection. Outstanding progress in computational prediction has achieved a significant contribution to the biomedical fields, including immunodiagnosis. In silico analysis may have an even more important role when information concerning antigens from etiologic agents of neglected diseases, such as leprosy, is scarce. The aim of this study was to provide mapping of B-cell epitopes from two Mycobacterium leprae-derived antigens (Ag85B and ML2055), confirm their antigenicity, and to assess the ability of in silico immunoinformatics tools to accurately predict them. Linear B-cell epitopes predicted by ABCpred and SVMTrip servers were compared to antigenic regions of synthetic overlapping peptides that exhibited reactivity to antibodies from patients with leprosy. Our in vitro results identified several immunodominant regions that had also been indicated by in silico prediction, providing agreement between experimental and simulated data. After chemical synthesis, we used enzyme-linked immunosorbent assays to determine the effectiveness of the first identified sequence (GTNVPAEFLENFVHG) which had 72 % sensitivity and 78 % specificity (AUC = 0.79) while the second one (PVSSEAQPGDPNAPS) had 72 % sensitivity and 93.8 % specificity (AUC = 0.85). Using dot blotting, an easy-to-read visual test, both peptides could distinguish sera from patients with leprosy from those with tuberculosis and from sera of healthy volunteers. Our findings suggest that these synthetic peptides, with some refinement, may be useful as serological diagnostic antigens for leprosy. In addition, it was displayed that immunoinformatics provides reliable information for mapping potential B-cell epitopes for development of peptide-based diagnostic assays for neglected diseases.


Assuntos
Antígenos de Bactérias/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Hanseníase/diagnóstico , Testes Sorológicos/métodos , Adulto , Anticorpos Antibacterianos/imunologia , Feminino , Humanos , Hanseníase/sangue , Hanseníase/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae
16.
Biologicals ; 73: 8-15, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34376341

RESUMO

Staphylococcus aureus is an important human opportunistic pathogen that can have a major influence on public health. Here, we aimed to evaluate different aspects of the immune response to a novel multi-epitope fusion protein (HMS) based on HlaH35L, MntC, and SACOL0723 proteins in comparison to the individual antigens. For this purpose, specific total IgG, IgG1, and IgG2a isotypes and the cytokines related to Th1, Th2, and Th17 were assessed. The Bio-efficiency of the fusion protein was evaluated by opsonic killing activity. The HMS fusion protein elicited a high specific IgG level and also induced a higher level of Th1, Th2, and Th17-related cytokines which were more polarized towards the Th1 and Th17 compared to individual antigens. The HMS-specific antisera also significantly promoted phagocytosis of S. aureus COL strain by mouse macrophages. In conclusion, the fusion protein might be an effective vaccine for potential protective immunity against a lethal infection of S. aureus in mice.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Citocinas/imunologia , Epitopos/imunologia , Imunoglobulina G/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Linfócitos T/imunologia
17.
Infect Immun ; 89(11): e0022421, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34370510

RESUMO

The immunomes of Ehrlichia chaffeensis and Ehrlichia canis have recently been revised to include immunodominant hypothetical proteins with conformational antibody epitopes. In this study, we examined 216 E. chaffeensis and 190 E. canis highly antigenic proteins according to ANTIGENpro and also performed a genome-wide hypothetical protein analysis (E. chaffeensis n = 104; E. canis n = 124) for immunoreactivity. Using cell-free protein expression and immunoanalysis, 118 E. chaffeensis and 39 E. canis proteins reacted with sera from naturally E. chaffeensis-infected patients or E. canis-infected dogs. Moreover, 22 E. chaffeensis and 18 E. canis proteins consistently and strongly reacted with a panel of patient or canine sera. A subset of E. chaffeensis (n = 18) and E. canis (n = 9) proteins were identified as immunodominant. Consistent with our previous study, most proteins were classified as hypothetical, and the antibody epitopes exhibited complete or partial conformation dependence. The majority (28/40, 70%) of E. chaffeensis and E. canis proteins contained transmembrane domains, and 19 (48%) were predicted to be secreted effectors. The antigenic repertoires of E. chaffeensis and E. canis were mostly diverse and suggest that the immunomes of these closely related ehrlichiae are dominated by species-specific conformational antibody epitopes. This study reveals a significant group of previously undefined E. chaffeensis and E. canis antigens and reaffirms the importance of conformation-dependent epitopes as targets of anti-Ehrlichia immune responses. These findings substantially expand our understanding of host-Ehrlichia immune responses, advance efforts to define the molecular features of protective proteins, and improve prospects for effective vaccines for the ehrlichioses.


Assuntos
Anticorpos Antibacterianos/imunologia , Ehrlichia canis/imunologia , Ehrlichia chaffeensis/imunologia , Epitopos/imunologia , Biologia Computacional , Ensaio de Imunoadsorção Enzimática , Humanos , Conformação Proteica
18.
PLoS One ; 16(8): e0250133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34437551

RESUMO

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.


Assuntos
Acetilglucosamina/imunologia , Infecções por Actinomycetales/veterinária , Anticorpos Antibacterianos/uso terapêutico , Proteínas de Bactérias/imunologia , Doenças dos Cavalos/prevenção & controle , Imunização Passiva/veterinária , Pneumonia Bacteriana/veterinária , Rhodococcus equi/imunologia , Infecções por Actinomycetales/imunologia , Infecções por Actinomycetales/microbiologia , Infecções por Actinomycetales/prevenção & controle , Animais , Animais Recém-Nascidos/imunologia , Animais Recém-Nascidos/microbiologia , Anticorpos Antibacterianos/imunologia , Feminino , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/microbiologia , Cavalos/imunologia , Cavalos/microbiologia , Imunização Passiva/métodos , Masculino , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/prevenção & controle
19.
Front Immunol ; 12: 696816, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305933

RESUMO

Early studies on vaccination of children with oncological diseases were only dedicated to the assessment of safety and immunogenicity of the drug. Mechanisms of the post-vaccination immune response were not investigated. This study involved 41 patients aged 7-15 years who were treated for solid tumors two or more years ago. Of these, 26 were vaccinated against diphtheria and tetanus with ADS-m toxoid. Fifteen children (i.e., controls) were not vaccinated. The vaccination tolerability and clinical characteristics of the underlying disease remission ware assessed. Lymphocyte subpopulations were investigated over time by flow cytometry at 1, 6, and 12 months. IgG anti-diphtheria and anti-tetanus toxoids levels were assessed by ELISA. Within the first day of the post-vaccination period, two (7.7%) children demonstrated moderate local reactions and increased body temperature (up to 38.0°C). Relapse and metastasis were not mentioned within a year after immunization. An increase in concentration of IgG antibodies, maintained for 12 months, were noted [2.1 (1.3-3.4) IU/ml against diphtheria (p <0.001), 6.4 (2.3-9.7) IU/ml against tetanus (p <0.001)]. In contrast to healthy children, those with a history of cancer demonstrated a decrease in the relative number of mature T lymphocytes, as well as in absolute number of cytotoxic T cells and B lymphocytes. In a month after the revaccination, a significant increase in absolute (p = 0.04) and relative (p = 0.007) numbers of T lymphocytes and T helpers was revealed. In a year, these values decreased to baseline levels. As for helpers, they decreased below baseline and control values (p = 0.004). In a year after the vaccination, there was a significant (p = 0.05) increase in lymphocyte level with a decrease in the number of NK cells and B cells as compared with controls. Revaccination against diphtheria and tetanus promoted proliferation of a total lymphocytic cell pool along with restoration of the T lymphocyte subpopulation in children with a history of solid tumors. The ADS-m toxoid has a certain nonspecific immunomodulatory effect. These findings are important, also in the midst of the coronavirus pandemic.


Assuntos
Imunidade Adaptativa/imunologia , Vacina contra Difteria e Tétano/imunologia , Neoplasias/imunologia , Vacinação , Adolescente , Anticorpos Antibacterianos/imunologia , Criança , Difteria/imunologia , Difteria/prevenção & controle , Vacina contra Difteria e Tétano/administração & dosagem , Humanos , Imunização Secundária , Subpopulações de Linfócitos/imunologia , Linfócitos/imunologia , Neoplasias/patologia , Federação Russa , Tétano/imunologia , Tétano/prevenção & controle
20.
Vet Microbiol ; 260: 109183, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34304027

RESUMO

Streptococcus suis serotype (cps) 1 and cps14 have been detected in association with severe diseases such as meningitis and polyarthritis in pigs. Though these two cps are very similar, only cps14 is an important zoonotic agent in Asia and only cps1 is described to be associated with diseases in suckling piglets rather than weaning piglets. The main objective of this study was to assess restriction of survival of cps14 and cps1 in porcine blood by IgG and IgM putatively cross-reacting with these two cps. Furthermore, we differentiate recent European cps1/14 strains by agglutination, cpsK sequencing, MLST and virulence-associated gene profiling. Our data confirmed cps1 of clonal complex 1 as an important pathotype causing polyarthritis in suckling piglets in Europe. The experimental design included also bactericidal assays with blood samples drawn at different ages of piglets naturally infected with different S. suis cps types including cps1 but not cps14. We report survival of a cps1 and a cps14 strain (both of sequence type 1) in blood of suckling piglets with high levels of maternal IgG binding to the bacterial surface. In contrast, killing of cps1 and cps14 was recorded in older piglets due to an increase of IgM as demonstrated by specific cleavage of IgM. Heterologous absorption of antibodies with cps1 or cps14 is sufficient to significantly increase the survival of the other cps. In conclusion, IgM elicited by natural S. suis infection is crucial for killing of S. suis cps1 and cps14 in older weaning piglets and has most likely the potential to cross-react between cps1 and cps14.


Assuntos
Anticorpos Antibacterianos/imunologia , Artrite/veterinária , Meningite/veterinária , Infecções Estreptocócicas/veterinária , Streptococcus suis/imunologia , Doenças dos Suínos/microbiologia , Animais , Artrite/microbiologia , Técnicas de Tipagem Bacteriana/veterinária , Reações Cruzadas , Meningite/microbiologia , Tipagem de Sequências Multilocus/veterinária , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus suis/patogenicidade , Suínos , Virulência , Desmame
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