RESUMO
Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
Assuntos
Imunidade nas Mucosas/imunologia , Interleucina-18/imunologia , Mucosa Intestinal/imunologia , Animais , Citocinas/imunologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/imunologia , Feminino , Células Caliciformes/imunologia , Interleucina-18/biossíntese , Mucosa Intestinal/metabolismo , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/imunologia , Ratos , Ratos Sprague-Dawley , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologiaRESUMO
Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
Assuntos
Pressão Hidrostática , Imunidade Inata , Canais Iônicos/metabolismo , Mecanotransdução Celular/imunologia , Animais , Endotelina-1/metabolismo , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/microbiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Transdução de SinaisRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.
Assuntos
Neutrófilos , Receptores de Fator Estimulador de Colônias de Granulócitos , Humanos , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos/genética , CitocinasRESUMO
The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.
Assuntos
Imunidade nas Mucosas/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , RNA Longo não Codificante/genética , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Colite/genética , Colite/imunologia , Colite/metabolismo , Imunidade nas Mucosas/efeitos dos fármacos , Interleucina-12/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Longo não Codificante/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Salmonella typhimurium/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Nine terpenoids were isolated from the leaves and flowers of Salvia amarissima, including a new acylated diterpenoid glucoside, amarisolide F (1), a new neo-clerodane diterpenoid, amarissinin D (2), which was isolated as an acetyl derivative (2a), and four known diterpenoids. The structure of amarisolide F (1) was elucidated by NMR and MS data analyses, as well as its methanolysis products 7 and 8, which also constituted new diterpenoids, named amarissinin E and 8- epi-amarissinin E, respectively. The absolute configuration of compound 7 was established by single-crystal X-ray diffraction. The cytotoxicity and anti-MDR effect of 1 in three phenotypes of the MCF-7 cell lines were assayed. Compound 1 was 2-3.6-fold more active than amarissinins A (3) and B (4), but several orders of magnitude less active than teotihuacanin (6) and reserpine.
Assuntos
Diterpenos/isolamento & purificação , Glucosídeos/química , Salvia/química , Acilação , Diterpenos/química , Diterpenos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Análise Espectral/métodosRESUMO
MAIN CONCLUSION : A RhoA-derived peptide fused to carrier molecules from plants showed enhanced biological activity of in vitro assays against respiratory syncytial virus compared to the RhoA peptide alone or the synthetic RhoA peptide. A RhoA-derived peptide has been reported for over a decade as a potential inhibitor of respiratory syncytial virus (RSV) infection both in vitro and in vivo and is anticipated to be a promising alternative to monoclonal antibody-based therapy against RSV infection. However, there are several challenges to furthering development of this antiviral peptide, including improvement in the peptide's bioavailability, development of an efficient delivery system and identification of a cost-effective production platform. In this study, we have engineered a RhoA peptide as a genetic fusion to two carrier molecules, either lichenase (LicKM) or the coat protein (CP) of Alfalfa mosaic virus. These constructs were introduced into Nicotiana benthamiana plants using a tobacco mosaic virus-based expression vector and targets purified. The results demonstrated that the RhoA peptide fusion proteins were efficiently expressed in N. benthamiana plants, and that two of the resulting fusion proteins, RhoA-LicKM and RhoA2-FL-d25CP, inhibited RSV growth in vitro by 50 and 80 %, respectively. These data indicate the feasibility of transient expression of this biologically active antiviral RhoA peptide in plants and the advantage of using a carrier molecule to enhance target expression and efficacy.
Assuntos
Proteínas de Plantas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/farmacologia , Vetores Genéticos , Testes de Sensibilidade Microbiana , Proteínas de Plantas/química , Proteínas de Plantas/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
MAIN CONCLUSION: The HRA2pl peptide expressed by transient transformation in N. tabacum plants is capable of inhibiting the binding of the human metapneumovirus to HEp-2 cells at the fusion stage. Human metapneumovirus (hMPV) is an agent responsible for acute respiratory infections that mainly affects children under 3 years, the elderly and immunocompromised patients. In children younger than 5 years, respiratory tract infections account for 20 % of deaths worldwide. However, there is currently no treatment or vaccine available against hMPV. The production of a safe, efficient and low cost treatment against this virus is a current challenge. Plants provide a system for recombinant protein production that is cost effective and is easier to scale up to an industrial level than other platforms; in addition, the plant tissue may be used as raw food, dried or, alternatively, proteins may be partially or fully purified and administered in aerosol or capsules as dry powder. In this study, we designed a gene expressing an antiviral peptide against hMPV based on the heptad repeat A domain of the F protein of the virus. We produced the recombinant peptide by a viral transient expression system (Magnifection(®)) in Nicotiana tabacum plants. The efficacy of this antiviral peptide was confirmed by in vitro assays in HEp-2 cell line. This is a promising result that can offer a prophylactic approach against hMPV.
Assuntos
Antivirais/química , Metapneumovirus/fisiologia , Nicotiana/genética , Peptídeos/farmacologia , Transformação Genética , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/farmacologia , Bioensaio , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Metapneumovirus/efeitos dos fármacos , Dados de Sequência Molecular , Infecções por Paramyxoviridae/patologia , Infecções por Paramyxoviridae/virologia , Peptídeos/química , Plantas Geneticamente Modificadas , Transformação Genética/efeitos dos fármacosRESUMO
Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.
Assuntos
Fármacos Anti-HIV/imunologia , Cloroplastos/genética , Proteína gp120 do Envelope de HIV/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos/administração & dosagem , Peptídeos/imunologia , Vacinas Sintéticas/administração & dosagem , Administração Oral , Animais , Fármacos Anti-HIV/administração & dosagem , Cloroplastos/imunologia , Feminino , Proteína gp120 do Envelope de HIV/genética , Soropositividade para HIV , Humanos , Imunidade nas Mucosas/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Peptídeos/genética , Plantas Geneticamente Modificadas , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Nicotiana/genéticaRESUMO
Extremophilic anaerobes are widespread in saline, acid, alkaline, and high or low temperature environments. Carbon is essential to living organisms and its fixation, degradation, or mineralization is driven by, up to now, six metabolic pathways. Organisms using these metabolisms are known as autotrophs, acetotrophs or carbon mineralizers, respectively. In anoxic and extreme environments, besides the well-studied Calvin-Benson-Bassham cycle, there are other five carbon fixation pathways responsible of autotrophy. Moreover, regarding carbon mineralization, two pathways perform this key process for carbon cycling. We might imagine that all the pathways can be found evenly distributed in microbial biotopes; however, in extreme environments, this does not occur. This manuscript reviews the most commonly reported anaerobic organisms that fix carbon and mineralize acetate in extreme anoxic habitats. Additionally, an inventory of autotrophic extremophiles by biotope is presented.
Assuntos
Acetatos/metabolismo , Bactérias Anaeróbias/metabolismo , Ciclo do Carbono , Dióxido de Carbono/metabolismo , Anaerobiose , Bactérias Anaeróbias/genética , Ecossistema , Redes e Vias Metabólicas , FilogeniaRESUMO
DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.
Assuntos
Toxinas Bacterianas/imunologia , Vacina contra Difteria, Tétano e Coqueluche/biossíntese , Exotoxinas/imunologia , Solanum lycopersicum/genética , Vacinas de Plantas Comestíveis/biossíntese , Animais , Anticorpos Antibacterianos/sangue , Bordetella pertussis/imunologia , Clostridium tetani/imunologia , Corynebacterium diphtheriae/imunologia , Difteria/prevenção & controle , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Epitopos/imunologia , Frutas/genética , Frutas/imunologia , Imunoglobulina G/sangue , Intestinos/imunologia , Pulmão/imunologia , Solanum lycopersicum/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Tétano/prevenção & controle , Vacinas de Plantas Comestíveis/imunologia , Coqueluche/prevenção & controleRESUMO
Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.
Assuntos
Toxinas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Enterotoxinas/metabolismo , Escherichia coli/metabolismo , Nicotiana/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Administração Oral , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Antígenos/imunologia , Sequência de Bases , Proteínas de Escherichia coli , Camundongos , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Tomato is one of the most important crops for human consumption. Its production is affected by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm), one of the most devastating bacterial pathogens of this crop. Several wild tomato species represent a source of natural resistance to Cmm. Here, we contrasted the transcriptomes of the resistant wild tomato species Solanum arcanum LA2157 and the susceptible species Solanum lycopersicum cv. Ailsa Craig, during the first 24 h of challenge with Cmm. We used three analyses approaches which demonstrated to be complementary: mapping to S. lycopersicum reference genome SL3.0; semi de novo transcriptome assembly; and de novo transcriptome assembly. In a global context, transcriptional changes seem to be similar between both species, although there are some specific genes only upregulated in S. arcanum during Cmm interaction, suggesting that the resistance regulatory mechanism probably diverged during the domestication process. Although S. lycopersicum showed enriched functional groups related to defense, S. arcanum displayed a higher number of induced genes related to bacterial, oomycete, and fungal defense at the first few hours of interaction. This study revealed genes that may contribute to the resistance phenotype in the wild tomato species, such as those that encode for a polyphenol oxidase E, diacyl glycerol kinase, TOM1-like protein 6, and an ankyrin repeat-containing protein, among others. This work will contribute to a better understanding of the defense mechanism against Cmm, and the development of new control methods.
Assuntos
Resistência à Doença/genética , Doenças das Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Transcriptoma , Infecções Bacterianas/microbiologia , Clavibacter , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genoma de Planta , Interações Hospedeiro-Patógeno , Fenótipo , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA-SeqRESUMO
Agave lechuguilla has one of the widest distributions among other agave species in the Chihuahuan Desert. Their capacity to grow in poorly developed soils and harsh conditions has been related to their association with plant growth-promoting rhizobacteria. In this work, we explored how soil properties and plant growth stage influence the composition of the rhizobacterial communities, their interactions, and the enzymatic activity and abundance of nitrogen-fixing bacteria and organic phosphorus-mineralizing bacteria in two subregions of the Chihuahuan Desert. We found that mature plants of lechuguilla stimulated the activity and abundance of nutrient-improvement rhizobacteria, and these soil samples had a higher content of total organic carbon, ammonium (NH4) and nitrite + nitrate (NO2+NO3). Nutrient availability seems to be an essential driver of the bacterial community's structure since the genera with more connections (hubs) were those with known mechanisms related to the availability of nutrients, such as env. OPS17 (Bacteroidetes), Gemmatimonadaceae uncultured, S0134terrestrial group, BD211terrestrial group (Gemmatimonadetes), Chthoniobacteracea and Candidatus Udaeobacter (Verrucomicrobia). This work shows that the late growth stages of lechuguilla recruit beneficial bacteria that favor its establishment and tolerance to harsh conditions of the arid lands.
Assuntos
Agave , Rizosfera , Bactérias/genética , Nutrientes , Solo , Microbiologia do SoloRESUMO
Bordetella pertusis causes whooping cough or pertussis, disease that has not been eradicated and is reemerging despite the availability and massive application for decades of vaccines, such as Boostrix® which is an acellular vaccine harboring two regions of S1 subunit of the pertussis toxin, one region of filamentous hemagglutinin and one region of pertactin. In 2008, the World Health Organization estimated 16 million new cases and 95% occurred in developing countries with 195,000 children's deaths. We attempt to improve the vaccine against whooping cough and reduce its production costs by obtaining plants and bacteria expressing a heterologous protein harboring pertactin, pertussis toxin, and filamentous hemagglutinin epitopes from B. pertussis and assessing its immunogenicity after oral administration to mice. First, we designed a synthetic gene that encodes a multiepitope, then it was cloned into a vector for transient transformation by infiltration of tobacco plants with low amounts of nicotine; the codon bias-optimized construct was also cloned into an Escherichia coli expression vector. Recombinant proteins from E. coli cells (PTF) and tobacco leaves (PTF-M3') were purified by nickel affinity with a yield of 0.740 mg of recombinant protein per g dry weight. Purified recombinant proteins were administered orally to groups of Balb/c mice using the Boostrix® vaccine and vehicle (PBS) as positive and negative controls, respectively. A higher mucosal and systemic antibody responses were obtained in mice receiving the PTF and PTF-M3' proteins than Boostrix® or PBS. These findings prove the concept that oral administration of multiepitope recombinant proteins expressed in plants may be a potential edible vaccine. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02107-1.
RESUMO
The need to produce food in a sustainable way to counteract the effects of excessive use of agrochemicals opens the door to the generation of new technologies that are not based on fossil fuels and are less toxic to ecosystems. Plant growth-promoting bacteria (PGPB) could represent an alternative to chemical biofertilizers and pesticides offering protection for biotic and abiotic stresses. In this work, a bacterial isolate from roots of castor bean (Ricinus communis) was identified and named as Bacillus cereus strain "Amazcala" (B.c-A). This isolate displayed the ability to solubilize inorganic phosphate and produce gibberellic acid (GA3). Moreover, this bacterium provided significant increases in height, stem width, dry weight, and total chlorophyll content in tomato plants. Interestingly, B.c-A also significantly decreased the severity of bacterial canker disease on tomato caused by Clavibacter michiganensis (Cmm) in preventive disease assays under greenhouse conditions. Based on our results, B.c-A can be considered as PGPB and a useful tool in Cmm disease control on tomato plant under greenhouse conditions.
RESUMO
It's been almost a century since immunologists started using adjuvants as tools to develop more effective vaccines. Despite the rising number of adjuvanted vaccines in the last decades, we still lack knowledge of the adjuvants' effects on antibody response. This study was aimed to test the effect of immunizing mice with the human Inactivated Influenza vaccine (IIV), either alone or combined with different widely used adjuvants on the specific antibody response induced. Differential levels of IgM and IgG subclasses were found with the different adjuvants tested. Higher levels of antibodies did not always correspond with a higher efficacy to interfere with the virus infectivity. Differences in neutralization properties are possibly mediated by the specificity of the repertoire of antibodies induced. The repertoire was studied using a phage display 7-mer peptide library to screen for epitopes/mimotopes recognized by serum pools from vaccinated mice. The selected phage clones included peptides that corresponded to conformational mimotopes since they have no homology with lineal sequences of the Influenza strains' proteins. Five peptides were identified as recognized by sera from mice immunized with the IIV vaccine alone, including peptides from the hemagglutinin stalk domain, and by sera from mice immunized with the vaccine plus the different adjuvants employed. Adjuvants elicited a more diverse repertoire of epitope-recognizing antibodies that recognized epitopes of the HA recombinant globular head. Mimotopes were theoretically located at the neutralizing antigenic sites of the globular head of Influenza A H1N1pdm09, Influenza A H3N2, and Influenza B hemagglutinin. This study illustrates how different adjuvants can modify the extent and quality of humoral immunity against the IIV vaccine and the effectiveness of vaccination.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra Influenza/imunologia , Potência de Vacina , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Biologia Computacional , Epitopos/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Biblioteca de Peptídeos , VacinaçãoRESUMO
Yersinia pestis is a pathogenic agent that causes the bubonic and pneumonic plague. The development of an efficient and low-cost oral vaccine against these diseases is highly desirable. In this study, the immunogenic fusion protein F1-V from Y. pestis was introduced into lettuce via Agrobacterium-mediated transformation, and putative transgenic lines were developed. The presence of the transgene in these putative transgenic lines was determined using polymerase chain reaction (PCR), and transgene integration and transgene copy number were confirmed following Southern blot analysis. The presence of specific F1-V transcripts was confirmed by reverse-transcriptase (RT)-PCR. Using monoclonal antibodies, ELISA and western blot analysis revealed that the expected antigenic F1-V protein was successfully expressed in transgenic lines. Mice immunized subcutaneously with lettuce expressing the F1-V antigen developed systemic humoral responses as 'proof of concept' of using lettuce as a production platform for the F1-V immunogen that could be used as a candidate plant-based vaccine against plague.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Lactuca/metabolismo , Vacina contra a Peste/imunologia , Vacina contra a Peste/metabolismo , Peste/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Yersinia pestis/imunologia , Animais , Antígenos de Bactérias/genética , Western Blotting , Ensaio de Imunoadsorção Enzimática , Lactuca/genética , Camundongos , Camundongos Endogâmicos BALB C , Peste/microbiologia , Vacina contra a Peste/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Rhizobium/genética , Yersinia pestis/patogenicidadeRESUMO
BACKGROUND: Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. RESULTS: A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. CONCLUSIONS: EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of their DNA-B component, short rep gene sequences located downstream to a CP-promoter-like domain. This assemblage of DNA-A-related sequences within the DNA-B IR is reminiscent of polyomavirus microRNAs and could be involved in the posttranscriptional regulation of the cognate viral rep gene, an intriguing possibility that should be experimentally explored.