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1.
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
2.
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
3.
Artigo em Inglês | MEDLINE | ID: mdl-34090612

RESUMO

Numbers of pathogenic bacteria can induce apoptosis in human host cells and modulate the cellular pathways responsible for inducing or inhibiting apoptosis. These pathogens are significantly recognized by host proteins and provoke the multitude of several signaling pathways and alter the cellular apoptotic stimuli. This process leads the bacterial entry into the mammalian cells and evokes a variety of responses like phagocytosis, release of mitochondrial cytochrome c, secretion of bacterial effectors, release of both apoptotic and inflammatory cytokines, and the triggering of apoptosis. Several mechanisms are involved in bacteria-induced apoptosis including, initiation of the endogenous death machinery, pore-forming proteins, and secretion of superantigens. Either small molecules or proteins may act as a binding partner responsible for forming the protein complexes and regulate enzymatic activity via protein-protein interactions. The bacteria induce apoptosis, attack the human cell and gain control over various types of cells and tissue. Since these processes are intricate in the defense mechanisms of host organisms against pathogenic bacteria and play an important function in host-pathogen interactions. In this chapter, we focus on the various bacterial-induced apoptosis mechanisms in host cells and discuss the important proteins and bacterial effectors that trigger the host cell apoptosis. The structural characterization of bacterial effector proteins and their interaction with human host cells are also considered.


Assuntos
Apoptose/imunologia , Bactérias , Infecções Bacterianas/imunologia , Proteínas de Bactérias , Fatores de Virulência , Bactérias/imunologia , Bactérias/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Humanos , Relação Estrutura-Atividade , Fatores de Virulência/química , Fatores de Virulência/imunologia
4.
Res Vet Sci ; 138: 100-108, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34126448

RESUMO

Strangles, which is caused by Streptococcus equi subspecies equi, is one of the most prevalent equine infectious diseases and poses heavy economic losses worldwide. Although various vaccines have been used for decades, they seemed to be sub-optimal to demonstrate effective protection, and the antigen component of vaccines against S. equi remains to be optimized. In the present study, three target antigens (M-like protein, α2-macroglobulin and IgG-binding protein, and glyceraldehyde-3-phosphate dehydrogenase) were selected and expressed. Mice were immunized and challenged, and their immune response and efficacy were evaluated. The results revealed that this optimized multi-antigen treatment elicited a high expression level of T-cell receptor, major histocompatibility complex I, toll-like receptor TLR-4, and increased specific antibody. In addition, the challenge experiment showed an evidently improved protection efficacy. The present work demonstrated that these three proteins might be used as a promising multicomponent subunit vaccine candidate against S. equi infection.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Infecções Estreptocócicas/prevenção & controle , Streptococcus/imunologia , Animais , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Streptococcus/enzimologia
5.
Nat Microbiol ; 6(6): 806-817, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958765

RESUMO

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.


Assuntos
Anopheles/microbiologia , Proteínas de Bactérias/imunologia , Lipase/imunologia , Mosquitos Vetores/microbiologia , Serratia/enzimologia , Serratia/isolamento & purificação , Animais , Anopheles/imunologia , Anopheles/parasitologia , Anopheles/fisiologia , Proteínas de Bactérias/genética , China , Feminino , Trato Gastrointestinal/microbiologia , Humanos , Lipase/genética , Malária Vivax/transmissão , Masculino , Mosquitos Vetores/imunologia , Mosquitos Vetores/parasitologia , Mosquitos Vetores/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium vivax/fisiologia , Serratia/genética , Serratia/fisiologia , Simbiose
6.
Int J Biol Macromol ; 183: 1346-1351, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34004200

RESUMO

Anti-idiotypic antibody technique is a new approach for the rapid development of insecticidal protein. In this study, anti-Cry1A polyclonal antibodies were used as antigen to screen the anti-idiotypic antibody that can simulate Cry1A toxins from a phage display human domain antibody (DAB) library. After four rounds of panning, five positive clones that have binding activities with anti-Cry1A polyclonal antibodies were obtained. Indirect competitive ELISA (IC-ELISA) results showed that the positive clone D6 showed significant inhibition for the binding of Cry1A toxins with anti-Cry1A polyclonal antibodies, and the inhibition ratio increased with the increase of D6 content. While, B3, F4, G5, C7 and the controls showed no obvious inhibition to Cry1A toxins. The results suggest that D6 is the "ß" subtype anti-idiotypic antibody, which can simulate Cry1A toxins and competitive binding with anti-Cry1A polyclonal antibodies. Meanwhile, D6 had certain binding activity with the brush border membrane vesicles (BBMV) of p. xylostella, which was the receptor of Cry1A toxins. The results of bioassay showed that D6 had certain insecticidal activity, and the lethal concentration of 50% (LC50) was 976 ng/cm2. This study provides basic materials and experience for the development of Cry toxin simulants.


Assuntos
Toxinas de Bacillus thuringiensis/imunologia , Endotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Biblioteca de Peptídeos , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos
7.
Res Vet Sci ; 137: 138-143, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33975192

RESUMO

Pasteurella multocida (P. multocida) infects the swine respiratory tract and mainly causes atrophic rhinitis (AR). Recently, many commercially inactivated and subunit vaccines have been used as preventive strategies. However, the best antigenic protein portion has not been selected, and the aluminum gel was used as the adjuvant, which may not induce full protection. P. multocida toxin (PMT) is the major virulence factor responsible for AR. PMT is a monomeric 146 kDa protein (approximately 1285 amino acids) encoded by the tox A gene. In this study, we expressed different fragments of recombinant PMT proteins, combined them with a water-in-oil-in-water adjuvant, and evaluated mice's immune response. The results indicated that the rPMT-C-immunized group showed significantly higher levels (p < 0.05) of IgG, IgG2a antibody and interferon-γ, IL-12 cytokine expression than other groups. Furthermore, vaccination with rPMT-C recombinant protein can provide homologous and heterologous protection against P. multocida challenge. In conclusion, our approach may be feasible for developing an effective subunit vaccine against atrophic rhinitis with a cost-down simple ingredient.


Assuntos
Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Pasteurella/veterinária , Pasteurella multocida , Rinite Atrófica/prevenção & controle , Adjuvantes Imunológicos , Animais , Imunização , Camundongos , Camundongos Endogâmicos ICR , Infecções por Pasteurella/imunologia , Infecções por Pasteurella/prevenção & controle , Rinite Atrófica/imunologia , Vacinas Sintéticas/uso terapêutico
8.
Life Sci ; 279: 119644, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34048813

RESUMO

Bacterial-derived extracellular vesicles could play a major role in attenuating and treating diseases. They play a major anti-infection role by modulating immune responses against pathogens and preventing infection by inhibiting pathogen localization and proliferation. In this study, outer membrane vesicles (ExHp-CD) released by Helicobacter pylori SS1 (H. pylori) and total antigens isolated from H. pylori SS1 (AgHp) were evaluated for their immunogenic potential and their effect on macrophage RAW 264.7 cells. Results demonstrated that both ExHp-CD and AgHp induced T helper 2 (Th2) immune response, which was reported to be important in immune protection against H. pylori infections. Both ExHp-CD and AgHp produced high levels of IL-10 and IL-4, while no significant levels of IL-12 p70 or IFN-γ were detected. However, ExHp-CD showed a better effect on macrophage RAW 264.7 cells compared to AgHp. Macrophage RAW 264.7 cells stimulated with 5, and 10 µg/mL of ExHp-CD showed an increased ratio of CD206 (M2 phenotype marker) and a decreased ratio of CD86 (M1 phenotype marker). Moreover, results suggested that the immunogenic effect that ExHp-CD possesses was attributed to their cargo of Epimerase_2 domain-containing protein (Epi_2D), Probable malate:quinone oxidoreductase (Pro_mqo), and Probable cytosol aminopeptidase (Pro_ca). Results demonstrated that ExHp-CD possesses an immunological activity to induce Th2 immune response against H. pylori infection with results comparable to AgHp. However, ExHp-CD showed higher efficacy regarding safety, biocompatibility, lack of toxicity, and hemocompatibility. Thus, it could serve as an immunogenic candidate with more desired characteristics.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vesículas Extracelulares/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Imunidade Celular/imunologia , Macrófagos/imunologia , Animais , Infecções por Helicobacter/imunologia , Interações Hospedeiro-Patógeno , Camundongos , Células RAW 264.7
9.
Fish Shellfish Immunol ; 114: 253-262, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33979691

RESUMO

Vibriosis, an illness caused by the Vibrio bacteria species, results in significant economic loss in olive flounder farms. Here we present a novel anti-Vibrio feed vaccine protecting multiple strains of Vibrio pathogens, a universal vaccine effect. The vaccine was generated by engineering Lactococcus lactis BFE920 to express the fusion antigens of Vibrio outer membrane protein K (OmpK) and flagellin B subunit (FlaB). These antigen genes are highly conserved among Vibrio species. Olive flounder (7.1 ± 0.8 g and 140 ± 10 g) were fed the vaccine adsorbed to a regular feed (1 × 107 CFU/g) for one week with a 1-week interval, repeating three times (a triple boost). The vaccinated fish increased the significant levels of antigen-specific antibodies, T cell numbers (CD4-1, CD4-2, and CD8α), cytokine production (T-bet and IFN-γ), and innate immune responses (TLR5M, IL-1ß, and IL-12p40). Also, the survival rates of adult and juvenile fish fed the vaccine were significantly elevated when challenged with V. anguillarum, V. alginolyticus, and V. harveyi. In addition, weight gain rate and feed conversion ratio were improved in vaccinated fish. The feed vaccine protected multiple Vibrio pathogens, a universal vaccine effect, by activating innate and adaptive immune responses. This oral vaccine may be developed as an anti-Vibrio vaccine to protect against a broad spectrum of Vibrio pathogens.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Linguado , Lactococcus lactis/metabolismo , Vibrioses/veterinária , Vibrio/metabolismo , Imunidade Adaptativa , Animais , Vacinas Bacterianas/administração & dosagem , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Imunidade Inata , Probióticos , Vibrio/imunologia , Vibrioses/prevenção & controle
10.
Nat Commun ; 12(1): 2574, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976140

RESUMO

Allergic asthma is characterized by elevated levels of IgE antibodies, type 2 cytokines such as interleukin-4 (IL-4) and IL-13, airway hyperresponsiveness (AHR), mucus hypersecretion and eosinophilia. Approved therapeutic monoclonal antibodies targeting IgE or IL-4/IL-13 reduce asthma symptoms but require costly lifelong administrations. Here, we develop conjugate vaccines against mouse IL-4 and IL-13, and demonstrate their prophylactic and therapeutic efficacy in reducing IgE levels, AHR, eosinophilia and mucus production in mouse models of asthma analyzed up to 15 weeks after initial vaccination. More importantly, we also test similar vaccines specific for human IL-4/IL-13 in mice expressing human IL-4/IL-13 and the related receptor, IL-4Rα, to find efficient neutralization of both cytokines and reduced IgE levels for at least 11 weeks post-vaccination. Our results imply that dual IL-4/IL-13 vaccination may represent a cost-effective, long-term therapeutic strategy for the treatment of allergic asthma as demonstrated in mouse models, although additional studies are warranted to assess its safety and feasibility.


Assuntos
Asma/terapia , Interleucina-13/antagonistas & inibidores , Interleucina-4/antagonistas & inibidores , Vacinação/métodos , Animais , Asma/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Doença Crônica/terapia , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intramusculares , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Camundongos , Camundongos Transgênicos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia
11.
Genes (Basel) ; 12(3)2021 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805671

RESUMO

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1ß and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.


Assuntos
Antibacterianos/farmacologia , Imunidade Inata/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/imunologia , Proteínas de Bactérias/imunologia , Células CACO-2 , Linhagem Celular Tumoral , Citocinas/imunologia , Humanos , Listeriose/tratamento farmacológico , Listeriose/imunologia , Testes de Sensibilidade Microbiana/métodos , Fatores de Virulência/imunologia
12.
Res Vet Sci ; 136: 422-429, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33812285

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase C (GapC) of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce a protective immune response against S. dysgalactiae infection. To investigate the immune response and protective efficacy induced by epitope-vaccines against S. dysgalactiae infection, we constructed epitope-vaccines GTB1, GB1B2, and GTB1B2 using a T cell epitope (GapC63-77, abbreviated as GT) and two B cell epitopes (GapC30-36, abbreviated as GB1, and GapC97-103, abbreviated as GB2), which were identified in GapC1-150 of S. dysgalactiae in tandem by a GSGSGS linker. BALB/c mice were immunized via an intramuscular injection with the epitope vaccines. The levels of the cytokines, IFN-γ, IL-4, and IL-17, secreted by splenic lymphocytes and the antibody levels in the sera of the immunized mice were detected by ELISA. The immunized mice were subsequently challenged with S. dysgalactiae, and the bacterial colonization in the immunized-mouse organs was examined using the plate counting method. The results showed that the level of the cytokines induced by GTB1B2 was lower than that induced by GapC1-150, but higher than that induced by other epitope vaccines. The level of IgG induced by GTB1B2 was lower than that induced by GapC1-150, but higher than the levels induced by other epitope vaccines. The bacterial colonization numbers in the organs of the mice immunized with GTB1B2 were higher those of the mice immunized with GapC1-150, but significantly lower than those from the mice immunized with other epitope-vaccines. Our results demonstrated that the T cell and B cell epitopes in the epitope-vaccines worked synergistically against bacterial challenge. The multi-epitope vaccine, GTB1B2, could induce stronger cellular and humoral immune responses, and provide a better protective effect against S. dysgalactiae infection.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Imunogenicidade da Vacina , Vacinas Estreptocócicas/imunologia , Streptococcus/imunologia , Animais , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C
13.
mBio ; 12(2)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879592

RESUMO

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.


Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Expressão Gênica , Genes Bacterianos , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/microbiologia
14.
Front Immunol ; 12: 619362, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33659004

RESUMO

Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210-269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αß-crystallin knockout cell line BoMac-cryab-, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab- induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.


Assuntos
Apoptose , Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Infecções por Mycoplasma/genética , Infecções por Mycoplasma/microbiologia , Mycoplasma bovis/fisiologia , Animais , Apoptose/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Genoma Bacteriano , Humanos , Ligantes , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/metabolismo
15.
BMC Vet Res ; 17(1): 123, 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33726780

RESUMO

BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. RESULTS: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. CONCLUSIONS: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.


Assuntos
Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina G/sangue , Mycoplasma hyopneumoniae/imunologia , Pneumonia Suína Micoplasmática/imunologia , Doenças dos Suínos/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Convalescença , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/imunologia , Pneumonia Suína Micoplasmática/sangue , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/sangue
16.
Food Chem ; 353: 129481, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-33725546

RESUMO

The interaction between conventional immunoglobulins (Igs) and the Ig-binding surface proteins of Staphylococcus aureus (S. aureus) have obstructed the development of immuno-assays to detect these bacteria. The current study aimed to select nanobodies (Nbs) recognizing specifically S. aureus and to establish an immuno-assay to uncover S. aureus contaminations in foods. An alpaca was immunized with an inactivated S. aureus strain followed by the construction of a Nb library from which four target-specific Nbs were retrieved. Subsequently, a sandwich ELISA employing the Nb147 and biotinylated-Nb147 pair to capture and to detect S. aureus, respectively, was established to possess a detection limit of 1.4 × 105 colony forming units (CFU)/mL. The dedicated immuno-assay has been verified by detecting 10 CFU/mL of S. aureus in milk samples after an 8 h-enrichment step. This study provides the basis of an easy, reproducible and effective immuno-assay to screen for S. aureus contaminations in foods.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Leite/microbiologia , Anticorpos de Domínio Único/imunologia , Staphylococcus aureus/isolamento & purificação , Animais , Proteínas de Bactérias/imunologia , Contaminação de Alimentos/análise , Limite de Detecção , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Staphylococcus aureus/metabolismo
17.
Front Immunol ; 12: 602689, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679740

RESUMO

An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1ß and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only the innate immune responses, but did not protect Salmo salar against P. salmonis. These results suggest that the knowledge of the formulation of vaccines based on P. salmonis proteins is useful as an effective therapy, this demonstrates the importance of the different research tools to improve the study of the different immune responses, resistance to diseases in the Atlantic salmon. We suggest that this vaccine can help prevent widespread infection by P. salmonis, in addition to being able to be used as a booster after a primary vaccine to maintain high levels of circulating protective antibodies, greatly helping to reduce the economic losses caused by the pathogen.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Peixes , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae , Salmo salar , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/prevenção & controle , Infecções por Piscirickettsiaceae/veterinária , Salmo salar/imunologia , Salmo salar/microbiologia
18.
Res Vet Sci ; 136: 303-309, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33744821

RESUMO

Pasteurella multocida is an important zoonotic pathogen that causes multiple diseases in both animals and humans. Test of good immunogenic proteins is beneficial for vaccine development and disease control. In the present study, we determined four novel immunogenic proteins of P. multocida by using 2-DE MALDI-TOF MS with immune serum. These four proteins included a trimethylamine-N-oxide reductase TorA, a translation elongation factor Ts, a phosphoglyceromutase PGAM, and a peroxiredoxin PrX. Among these proteins, TorA, Prx, and PGAM were successfully expressed by using E. coli. Western-blotting assays showed that recombinant TorA, Prx, and/or PGAM displayed good reactions with infectious sera of P. multocida serogroups A, B, D and F. Immunization of either rTorA, rPrx, and/or rPGAM induced significantly high levels of antibodies as well as IFN-γ, IL-4 and IL-10 in mice (P < 0.01). Protective efficacy tests revealed that vaccination of either rTorA, rPrx, and/or rPGAM protected 60% ~ 80% of the tested mice against the challenge with P. multocida field isolate. Our results obtained from the present study suggest that these three proteins could be tested as good vaccine candidates against P. multocida infections.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Imunização Passiva/veterinária , Pasteurella multocida/imunologia , Animais , Eletroforese em Gel Bidimensional/veterinária , Soros Imunes/imunologia , Espectrometria de Massas/veterinária , Camundongos , Infecções por Pasteurella/imunologia , Infecções por Pasteurella/veterinária , Suínos , Doenças dos Suínos/microbiologia
19.
Medicine (Baltimore) ; 100(8): e24615, 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33663071

RESUMO

ABSTRACT: The T-SPOT.TB assay detects cellular immune responses to 2 core Mycobacterium tuberculosis antigens, early secreted antigenic target of 6-kDa protein (ESAT-6) and culture filtrate protein-10 (CFP-10). T-SPOT.TB has been recently used for auxiliary diagnosis of active pulmonary tuberculosis (PTB). However, testing can produce inconsistent results due to differential PTB patient immune responses to these antigens, prompting us to identify factors underlying inconsistent results.Data were retrospectively analyzed from 1225 confirmed PTB patients who underwent T-SPOT.TB testing at 5 specialized tuberculosis hospitals in China between December 2012 and November 2015. Numbers of spot-forming cells (SFCs) reflecting T cell responses to ESAT-6 and CFP-10 antigens were recorded then analyzed via multivariable logistic regression to reveal factors underlying discordant T cell responses to these antigens.The agreement rate of 84.98% (82.85%-86.94%) between PTB patient ESAT-6 and CFP-10 responses demonstrated high concordance. Additionally, positivity rates were higher for ESAT-6 than for CFP-10 (84.8% vs 80.7%, P < .001), with ESAT-6 and CFP-10 microwell SFC numbers for each single positive group not differing significantly (P > .99), while spot numbers of the single positive group were lower than numbers for the double positive group (P < .001). Elderly patients (aged ≥66 years) and patients receiving retreatment were most likely to have discordance results.ESAT-6 promoted significantly more positive T-SPOT.TB results than did CFP-10 in PTB patients. Advanced age and retreatment status were correlated with discordant ESAT-6 and CFP-10 results. Assessment of factors underlying discordance may lead to improved PTB diagnosis using T-SPOT.TB.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Humanos , Imunidade Celular/imunologia , Testes Imunológicos/normas , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
20.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33741626

RESUMO

Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonization of mucosa-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate Ligilactobacillus agilis La3 (previously Lactobacillus agilis) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of L. agilis La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed to facilitate rapid selectable integration of expression cassettes into the L agilis La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a Clostridium perfringens antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, L agilis La3::P eft -rnetB and L agilis La3::P cwah -rnetB, were constructed and characterized, and they showed potential for future application as live vaccines in chickens.IMPORTANCE Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection generally has not proven effective for mucosa-dwelling microorganisms that live within the gastrointestinal tract. Utilizing live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, Ligilactobacillus agilis La3, an especially effective gut colonizer, has been analyzed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen Clostridium perfringens was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools and novel constitutive expression systems and further development of L. agilis La3 as a live delivery vehicle for recombinant proteins.


Assuntos
Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Expressão Gênica/imunologia , Genoma Bacteriano , Lactobacillus/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Clostridium perfringens/fisiologia , Lactobacillus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Atenuadas/imunologia
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