RESUMO
Introduction: The intestinal immune system plays a pivotal role in the induction of immune responses against food. In the case of T cell response, dendritic cells (DCs) are especially important. However, the regulation of immune responses to food by intestinal DCs has been poorly described. In this study, we analyzed the effect of Lactococcus lactis subsp. cremoris YRC3780, a lactic acid bacterial strain isolated from kefir, a traditional fermented milk product, on the immune responses induced by antigen presentation by intestinal DCs to T cells as well as the mechanism of action of these immunomodulatory effects. It has been shown that L. cremoris YRC3780 ameliorates the symptoms of pollinosis in both animal and human studies. Methods: CD11c+ cells from mesenteric lymph nodes (MLNs) of BALB/c mice were cultured as MLN DCs with L. cremoris YRC3780 and expression of genes inducing regulatory T cells (Tregs) was examined by qPCR. In addition, MLN DCs were cocultured with CD4+ T cells from DO11.10 transgenic mice expressing an ovalbumin (OVA)-specific TCR and the OVA antigen peptide and L. cremoris YRC3780. Induction of Tregs was examined by flow cytometry, gene expression was analyzed by DNA microarray and qPCR, and the production of cytokines was measured by ELISA. MLN DCs from TLR2-deficient mice and components of L. cremoris YRC3780 were used to examine the recognition of YRC3780 by MLN DCs. Results: L. cremoris YRC3780 enhanced the expression of genes involved in Treg induction in MLN DCs and induced Foxp3+CD4+T cells in an MLN DC and CD4+ T-cell co-culture system. The effect on MLN DCs was likely mediated by receptors other than TLR2. Together with microarray analyses of CD4+ T cell gene expression and cytokine ELISA, it was demonstrated that L. cremoris YRC3780 promoted the induction of Th1 and Tregs, and regulated the balance of Th1/Th2 and Treg/Th17 cells involving multiple genes via the antigen-presentation of MLN DCs. Discussion: Our findings provide insights into the modulation of intestinal immune responses mediated by DCs and the antiallergic effects of lactic acid bacteria.
Assuntos
Diferenciação Celular , Células Dendríticas , Lactococcus lactis , Linfonodos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Células Dendríticas/imunologia , Camundongos , Linfonodos/imunologia , Lactococcus lactis/imunologia , Diferenciação Celular/imunologia , Mesentério/imunologia , Citocinas/metabolismo , Camundongos Transgênicos , Ativação Linfocitária/imunologia , Técnicas de Cocultura , FemininoRESUMO
Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: ⢠Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. ⢠The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. ⢠Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.
Assuntos
Galinhas , Microbioma Gastrointestinal , Lactococcus lactis , Ligante RANK , Proteínas Recombinantes , Animais , Galinhas/imunologia , Administração Oral , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Lactococcus lactis/imunologia , Ligante RANK/imunologia , Ligante RANK/genética , Ligante RANK/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/administração & dosagem , Infecções por Birnaviridae/prevenção & controle , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Vírus da Doença Infecciosa da Bursa/imunologia , Vírus da Doença Infecciosa da Bursa/genética , Diferenciação Celular , Nódulos Linfáticos Agregados/imunologiaRESUMO
Brucella infections typically occur in mucosal membranes, emphasizing the need for mucosal vaccinations. This study evaluated the effectiveness of orally administering Lactococcus lactis (L. lactis) for producing the Brucella abortus multi-epitope OMPs peptide. A multi-epitope plasmid was generated through a reverse vaccinology method, and mice were administered the genetically modified L. lactis orally as a vaccine. The plasmid underwent digestion, synthesizing a 39 kDa-sized protein known as OMPs by the target group. The sera of mice that were administered the pNZ8124-OMPs-L. lactis vaccine exhibited a notable presence of IgG1 antibodies specific to outer membrane proteins (OMPs), heightened levels of interferon (IFN-λ) and tumor necrosis factor alpha (TNF-α), and enhanced transcription rates of interleukin 4 (IL-4) and interleukin 10 (IL-10). The spleen sections from the pNZ8124-OMPs-L. lactis and IRIBA group had less morphological damage associated with inflammation, infiltration of lymphocytes, and lesions to the spleen. The findings present a novel approach to utilizing the food-grade, non-pathogenic L. lactis as a protein cell factory to synthesize innovative immunological candidate OMPs. This approach offers a distinctive way to evaluate experimental medicinal items' practicality, safety, affordability, and long-term sustainability.
Assuntos
Vacina contra Brucelose , Brucella abortus , Brucelose , Lactococcus lactis , Camundongos Endogâmicos BALB C , Animais , Brucella abortus/imunologia , Brucelose/prevenção & controle , Brucelose/imunologia , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Vacina contra Brucelose/imunologia , Vacina contra Brucelose/administração & dosagem , Vacina contra Brucelose/genética , Camundongos , Feminino , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Epitopos/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Baço/imunologia , Vetores Genéticos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Citocinas/metabolismoRESUMO
Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.
Assuntos
Ciclídeos , Doenças dos Peixes , Infecções Estreptocócicas , Streptococcus agalactiae , Animais , Streptococcus agalactiae/imunologia , Infecções Estreptocócicas/veterinária , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/imunologia , Doenças dos Peixes/prevenção & controle , Doenças dos Peixes/imunologia , Ciclídeos/imunologia , Administração Oral , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Estreptocócicas/imunologia , Vacinas Estreptocócicas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genéticaRESUMO
Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines.
Assuntos
Adjuvantes Imunológicos , Flagelina , Lactococcus lactis , Camundongos Endogâmicos BALB C , Ovalbumina , Receptor 5 Toll-Like , Lactococcus lactis/imunologia , Animais , Flagelina/imunologia , Flagelina/administração & dosagem , Camundongos , Humanos , Ovalbumina/imunologia , Ovalbumina/administração & dosagem , Receptor 5 Toll-Like/imunologia , Adjuvantes Imunológicos/administração & dosagem , Feminino , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Imunização/métodos , Administração IntranasalRESUMO
BACKGROUND: The development of an influenza vaccine for poultry that provides broadly protective immunity against influenza H5Nx viruses is a challenging goal. RESULTS: Lactococcus lactis (L. lactis)/pNZ8149-HA1-M2 expressing hemagglutinin-1 (HA1) of A/chicken/Vietnam/NCVD-15A59/2015 (H5N6) and the conserved M2 gene of A/Vietnam/1203/2004 (H5N1) was generated. L. lactis/pNZ8149-HA1-M2 could induce significant humoral, mucosal and cell-mediated immune responses, as well as neutralization antibodies. Importantly, L. lactis/pNZ8149-HA1-M2 could prevent disease symptoms without significant weight loss and confer protective immunity in a chicken model against lethal challenge with divergent influenza H5Nx viruses, including H5N6 and H5N1. CONCLUSIONS: L. lactis/pNZ8149-HA1-M2 can serve as a promising vaccine candidate in poultry industry for providing protection against H5Nx virus infection in the field application.
Assuntos
Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza/imunologia , Lactococcus lactis , Infecções por Orthomyxoviridae/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Animais , Anticorpos Antivirais , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/imunologia , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Vacinação/veterinária , Vacinas Sintéticas/imunologiaRESUMO
Dengue fever (DF) is a mosquito-borne disease still with no effective treatment or vaccine available. A randomized, placebo-controlled, double-blinded, parallel-group trial was undertaken to evaluate the efficacy of oral intake of Lactococcus lactis strain plasma (LC-Plasma) on the presentation and severity of DF-like symptoms among healthy volunteers. Study participants (320) were assigned into two groups, and consumed either placebo or LC-Plasma tablets (approximately 100 billion cells/day) for 8 weeks. The clinical symptoms of DF were self-recorded through questionnaires, and exposure to DENV was determined by serum antibody and/or DENV antigen tests. No significant differences between groups were observed for exposure to DENV, or the symptomatic ratio. Results obtained showed that participants from the LC-Plasma group reported a significant reduction in the cumulative incidence days of DF-like symptoms, which include fever (p < 0.001), muscle pain (p < 0.005), joint pain (p < 0.001), and pain behind the eyes (p < 0.001), compared to that of the placebo group. Subgroup analysis revealed a significantly (p < 0.05) reduced severity score in the LC-Plasma group when study sites were separately analyzed. Overall, our findings suggest that LC-Plasma supplementation reduces the cumulative days with DF-like symptoms, and the severity of the symptoms. Daily oral intake of LC-Plasma, hence, is shown to mitigate the DF-like symptoms.
Assuntos
Vírus da Dengue/imunologia , Dengue/prevenção & controle , Lactococcus lactis/imunologia , Probióticos/administração & dosagem , Administração Oral , Adulto , Dengue/epidemiologia , Dengue/virologia , Método Duplo-Cego , Feminino , Humanos , Malásia/epidemiologia , Masculino , Resultado do TratamentoRESUMO
Foot and mouth disease virus (FMDV), whose transmission occurs through mucosal surfaces, can also be transmitted through aerosols, direct contact, and pollutants. Therefore, mucosal immunity can efficiently inhibit viral colonization. Since vaccine material delivery into immune sites is important for efficient oral mucosal vaccination, the M cell-targeting approach is important for effective vaccination given M cells are vital for luminal antigen influx into the mucosal lymph tissues. In this study, we coupled M cell-targeting ligand Co1 to multi-epitope TB1 of FMDV to obtain TB1-Co1 in order to improve delivery efficiency of the multi-epitope protein antigen TB1. Lactococcus lactis (L. lactis) was engineered to express heterologous antigens for applications as vaccine vehicles with the ability to elicit mucosal as well as systemic immune responses. We successfully constructed L. lactis (recombinant) with the ability to express multi-epitope antigen proteins (TB1 and TB1-Co1) of the FMDV serotype A (named L. lactis-TB1 and L. lactis-TB1-Co1). Then, we investigated the immunogenic potential of the constructed recombinant L. lactis in mice and guinea pigs. Orally administered L. lactis-TB1 as well as L. lactis-TB1-Co1 in mice effectively induced mucosal secretory IgA (SIgA) and IgG secretion, development of a strong cell-mediated immune reactions, substantial T lymphocyte proliferation in the spleen, and upregulated IL-2, IFN-γ, IL-10, and IL-5 levels. Orally administered ligand-conjugated TB1 promoted specific IgG as well as SIgA responses in systemic and mucosal surfaces, respectively, when compared to orally administered TB1 alone. Then, guinea pigs were orally vaccinated with L. lactis-TB1-Co1 plus adjuvant CpG-ODN at three different doses, L. lactis-TB1-Co1, and PBS. Animals that had been immunized with L. lactis-TB1-Co1 plus adjuvant CpG-ODN and L. lactis-TB1-Co1 developed elevated antigen-specific serum IgG, IgA, neutralizing antibody, and mucosal SIgA levels, when compared to control groups. Particularly, in mice, L. lactis-TB1-Co1 exhibited excellent immune effects than L. lactis-TB1. Therefore, L. lactis-TB1-Co1 can induce elevations in mucosal as well as systemic immune reactions, and to a certain extent, provide protection against FMDV. In conclusion, M cell-targeting approaches can be employed in the development of effective oral mucosa vaccines for FMDV.
Assuntos
Epitopos/imunologia , Vírus da Febre Aftosa/metabolismo , Febre Aftosa/imunologia , Lactococcus lactis/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/sangue , Formação de Anticorpos , Modelos Animais de Doenças , Feminino , Vírus da Febre Aftosa/genética , Cobaias , Imunidade nas Mucosas/imunologia , Imunização , Imunoglobulina A Secretora , Lactococcus lactis/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Vacinação , Vacinas Virais/imunologiaRESUMO
In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.
Assuntos
Infecções por Birnaviridae/prevenção & controle , Proteínas de Peixes/metabolismo , Imunidade Inata , Vírus da Necrose Pancreática Infecciosa/patogenicidade , Interferon Tipo I/metabolismo , Lactococcus lactis/metabolismo , Probióticos , Salmo salar/microbiologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/microbiologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Proteínas de Peixes/genética , Pesqueiros , Interações Hospedeiro-Patógeno , Vírus da Necrose Pancreática Infecciosa/crescimento & desenvolvimento , Vírus da Necrose Pancreática Infecciosa/imunologia , Interferon Tipo I/genética , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Salmo salar/genética , Salmo salar/imunologia , Salmo salar/virologia , Carga Viral , eIF-2 Quinase/metabolismoRESUMO
Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-ß, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naïve T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-ß in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance.
Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Lactococcus lactis/imunologia , Probióticos/administração & dosagem , Administração Oral , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Glicemia/análise , Linfócitos T CD4-Positivos , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Feminino , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Proteínas de Fímbrias/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Linfonodos/citologia , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Baço/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.
Assuntos
Proteínas de Peixes/metabolismo , Infecções por Flavobacteriaceae/prevenção & controle , Flavobacterium/patogenicidade , Interferon gama/metabolismo , Lactococcus lactis/metabolismo , Oncorhynchus mykiss/microbiologia , Probióticos/administração & dosagem , Administração Oral , Animais , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/metabolismo , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/imunologia , Interações Hospedeiro-Patógeno , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/metabolismo , FilogeniaRESUMO
Background: Cystic echinococcosis is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods: The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results: Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. Mucosal IgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion: Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice.
Assuntos
Antígenos de Helmintos/imunologia , Echinococcus granulosus/imunologia , Proteínas de Helminto/imunologia , Imunidade Humoral/imunologia , Fenômenos Imunogenéticos/fisiologia , Lactococcus lactis/imunologia , Animais , Antígenos de Helmintos/administração & dosagem , Feminino , Proteínas de Helminto/administração & dosagem , Imunidade Humoral/efeitos dos fármacos , Fenômenos Imunogenéticos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologiaRESUMO
AIMS: Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response pattern. The development of nonspecific immunotherapy is one of the primary goals for the control of this disease. METHODS AND RESULTS: In this study, we evaluated the therapeutic effects of Lactococcus lactis-producing mycobacterial heat shock protein 65 (LLHsp65) in an ovalbumin (OVA)-induced allergic asthma model. OVA-challenged BALB/c mice were orally administrated with LLHsp65 for 10 consecutive days. The results demonstrate that LLhsp65 attenuates critical features of allergic inflammation, like airway hyperresponsiveness and mucus production. Likewise, the treatment decreases the pulmonary eosinophilia and the serum level of OVA-specific IgE. In addition to deviating immune responses towards Th1-cytokine profile, increase regulatory T cells, and cytokine levels, such as IL-6 and IL-10. CONCLUSIONS: Our results reveal that the mucosal immunotherapy of LLHsp65 significantly reduces the overall burden of airway allergic inflammation, suggesting a promising therapeutic strategy for allergic asthma treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This research reveals new perspectives on nonspecific immunotherapy based on the delivery of recombinant proteins by lactic acid bacteria to treat of allergic disorders.
Assuntos
Asma/tratamento farmacológico , Proteínas de Bactérias/farmacologia , Chaperonina 60/farmacologia , Inflamação/tratamento farmacológico , Lactococcus lactis/imunologia , Administração Oral , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hipersensibilidade/tratamento farmacológico , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoterapia , Lactococcus lactis/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Linfócitos T Reguladores/imunologiaRESUMO
Since Brucella infection mostly occurs through the mucosal surfaces, immune response induced by vaccine that is delivered by a way of mucosal route can be drastically enhanced to control the brucellosis. Omp31is the major outer membrane protein of Brucella, and is considered as a protective antigen against Brucella infection. Accordingly, Lactococcus lactis has been used as an antigen-delivering vector to develop a vaccine-induced mucosal response for having a safer vaccination against brucellosis. A designed omp31 gene fused to the usp45 signal peptide and M6 cell wall anchor was sub cloned in the pNZ7021 expression vector, and a recombinant L. lactis displaying Omp31 was constructed. Omp31 protein expression was confirmed using Western blotting and immunofluorescence analysis. Animals were orally and intraperitoneally immunized with live or killed L. lactis expressing Omp31, respectively. The humoral and cellular immune responses were evaluated by measuring the specific cytokines and antibodies. sIgA, serum IgA, IgM, and total IgG antibodies significantly increased in the mice immunized with live recombinant L. lactis expressing Omp31 and also serum IgM, and total IgG antibodies significantly increased in mice immunized with killed recombinant L. lactis expressing Omp31. Among IgG subtypes, IgG2a response was significantly higher in both groups compared to IgG1. In mice groups immunized with recombinant L. lactis, the IFN-γ and IL-10 level elevated; however, there was no change in the level of IL-4. These results indicated that recombinants L. lactis induce both humoral and cellular immune responses in mice, and also vaccines based on L. lactis-derived live carriers are promising interventions against Brucella melitensis infections.
Assuntos
Proteínas da Membrana Bacteriana Externa , Vacina contra Brucelose , Brucella melitensis/genética , Brucelose , Lactococcus lactis , Microrganismos Geneticamente Modificados , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacina contra Brucelose/genética , Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Feminino , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/imunologiaRESUMO
BACKGROUND: The microbiome is now known for its important role in whole-body homeostasis. A dysbiosis of the normal microbiota is correlated with metabolic disorders. In this sense, the search for compounds able to modulate the microbiome is needed. Resveratrol, a natural compound found in grapes seems to be a promising candidate. OBJECTIVE: In this study, our motivation was to evaluate the effects of the association between Resveratrol and Lactococcus lactis, a probiotic, on the composition of the gastrointestinal microbiota and body weight of mice. METHODS: Twenty female mice were divided into 4 groups: (1) standard diet, (2) standard diet plus Lactococcus lactis, (3) standard diet plus resveratrol, and (4) standard diet plus Lactococcus lactis and resveratrol. At the end of the treatment period, samples of blood, mucus, stomach, and small and large intestines were collected for analysis. Total levels of Immunoglobulin A and Immunoglobulin E, Lac+ and Lac- bacteria and Lactobacillus were measured. RESULTS: The main results indicate that the association between resveratrol and probiotics was able to decrease mice body weight, as compared to the other groups, in addition to decrease the number of Lac- bacteria and increasing the number of Lac+ bacteria. The levels of secretory IgA were also decreased, compared to the animals treated with only probiotics or resveratrol. CONCLUSION: We observed potential synergism between Resveratrol and Lactococcus lactis mainly in modulating the stomach and intestinal microbiota.
Assuntos
Peso Corporal/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Lactococcus lactis/imunologia , Probióticos/administração & dosagem , Resveratrol/administração & dosagem , Animais , Peso Corporal/imunologia , Dieta/métodos , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/imunologia , Feminino , Microbioma Gastrointestinal/imunologia , Imunoglobulina A/biossíntese , Imunoglobulina E/sangue , Intestino Grosso/efeitos dos fármacos , Intestino Grosso/imunologia , Intestino Grosso/microbiologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Estômago/efeitos dos fármacos , Estômago/imunologia , Estômago/microbiologiaRESUMO
Most of the current approaches to influenza vaccine design focus on antibodies against influenza (HA). However, these influenza vaccines typically provide strain-specific protection against mostly homologous subtypes. There is an urgent need to develop a universal vaccine that confers cross-protection against influenza viruses. Of note, the HA stalk domain (HAsd) is a promising target for such an influenza vaccine. In this study, we generated recombinant Lactococcus lactis (L. lactis)/pNZ8150-phosphatidylglycerophosphate synthetase A (pgsA)-HAsd, in which pgsA was used as an anchor protein, and investigated the immunogenicity of HAsd in a mouse model by oral administration without the use of a mucosal adjuvant. Compared with L. lactis/pNZ8150-pgsA, mice were orally vaccinated with L. lactis/pNZ8150-pgsA-HAsd and then produced strong humoral and mucosal immune responses. Importantly, L. lactis/pNZ8150-pgsA-HAsd provided cross-protection against H5N1, H3N2 and H1N1 virus infections. Our data support the hypothesis that HAsd presented on the surface of L. lactis can provide cross-protective immunity against divergent influenza A viruses. Taken together, these findings suggest that L. lactis/pNZ8150-pgsA-HAsd can be considered an alternative approach to developing a novel universal vaccine during an influenza A pandemic. Abbreviations: HA, HAsd, HA stalk domain; L. lactis, Lactococcus lactis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IFA, immunofluorescence assay; PBS, phosphate-buffered saline; pgsA, phosphatidylglycerophosphate synthetase A; SPF, specific pathogen-free; CFU, colony-forming unit; BSL-3, biosafety level-3 laboratory; TCID50, 50% tissue culture infective dose; ELISA, enzyme-linked immunosorbent assay; OD, optical density; LTB, liable enterotoxin B subunit; CTB, cholera toxin B subunit.
Assuntos
Proteção Cruzada/imunologia , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Vírus da Influenza A/imunologia , Lactococcus lactis/genética , Infecções por Orthomyxoviridae/imunologia , Administração Oral , Animais , Anticorpos Antivirais/sangue , Feminino , Hemaglutininas Virais/administração & dosagem , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Lactococcus lactis/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20 µl of functional ice cream with 1012 CFU/ml of r-L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN-γ and TGF-ß cytokines increased in the 20 µl ice cream treatment group as well as 40 µg/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20 µl ice cream with 1012 CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses.
Assuntos
Dessensibilização Imunológica/métodos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Sorvetes , Lactococcus lactis/imunologia , Probióticos/administração & dosagem , Alérgenos/genética , Alérgenos/imunologia , Animais , Anticorpos/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Biomarcadores , Clonagem Molecular , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Hipersensibilidade/metabolismo , Imunização , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Lactococcus lactis/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Pólen/genética , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Spring viremia of carp virus (SVCV) causes devastating disease in aquaculture, resulting in significant economic impact. To develop an effective means against SVCV infection, a Lactococcus lactis (L.lactis) based subunit vaccine (pNZ-UGA) was developed based on surface displaying of SVCV glycoprotein using anchoring motif of the cA (C terminus of the peptidoglyvsn-binding) domains of AcmA, a major autolysin from L.lactis. The surface expression of SVCV glycoprotein was verified by indirect immunofluorescence assay. The efficacy of the constructed vaccine was further evaluated in common carp. The results showed that the higher levels of specific IgM could be detected in fish vaccinated with pNZ-UGA, compared with that in PBS and L.lactis groups. Immune-related genes including TNF-α, IL-6b, IL-1ß, Cxcr 1, Cxca, IFNg2b, I-IFN, and IgM expression in pNZ-UGA group were strongly up-regulated, revealing that robust innate immune response was induced. Notably, the lowest cumulative mortality (13.46%) was observed in fish vaccinated with pNZ-UGA vaccine after SVCV challenge, whereas the cumulative mortality were 100.00% and 92.31% in PBS and L.lactis groups, respectively. This study suggests the potential use of the recombinant L.lactis with surface displaying antigen proteins as effective vaccines against SVCV and other fish virus infection.
Assuntos
Carpas/imunologia , Doenças dos Peixes/prevenção & controle , Glicoproteínas/imunologia , Lactococcus lactis/imunologia , Infecções por Rhabdoviridae/prevenção & controle , Rhabdoviridae/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Carpas/sangue , Carpas/virologia , Citocinas/genética , Citocinas/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Lactococcus lactis/genética , Infecções por Rhabdoviridae/veterináriaRESUMO
Some lactic acid bacteria (LAB) are known to improve atopic dermatitis (AD) through the regulation and stimulation of the host immune system. In this study, we found that ingestion of yogurt containing Lactococcus lactis 11/19-B1 strain (L. lactis 11/19-B1) daily for 8 weeks significantly improved the severity scoring of atopic dermatitis (SCORAD) system score from 38.8 ± 14.4 to 24.2 ± 12.0 in children suffering from AD. We tried to identify which LAB species among the five species contained in the test yogurt contributed to the improvement in AD pathology using an AD mouse model induced by repeated application of 1-fluoro-2, 4-dinitrobenzene (DNFB). AD-like skin lesions on the dorsal skin and ear were most improved by L. lactis 11/19-B1 intake among the five LAB species. In addition, analysis of CD4+ T cell subsets in Peyer's patches (PPs) and cervical lymph nodes (CLNs) indicated that the intake of L. lactis 11/19-B1 generally suppressed all subsets related to inflammation, i.e., Th1, Th2 and Th17, instead of activating the suppressive system, Treg, in the AD mouse model. Histological observations showed ingestion of L. lactis 11/19-B1 significantly suppressed severe inflammatory findings, such as inflammatory cell filtration, epidermal erosion and eosinophil infiltration. These results suggest that the immunomodulatory effects of L. lactis 11/19-B1 contribute to improvements in AD pathology.
Assuntos
Dermatite Atópica , Lactococcus lactis/imunologia , Pele , Iogurte , Adolescente , Animais , Criança , Pré-Escolar , Dermatite Atópica/dietoterapia , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/patologia , Pele/imunologia , Pele/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
Most pathogens initiate infection via the mucosa, therefore delivery of vaccines directly to the mucosa is likely to be advantageous for stimulating protective immunity at the site of entry. PilVax is a novel mucosal vaccine platform that harnesses Lactococcus lactis bacteria engineered to stably express multiple copies of vaccine peptide antigens within pili, hair-like structures which extend from the cell wall. This strategy elicited systemic and mucosal antibody responses to a model antigen after intranasal immunization, but has not been tested for its capacity to stimulate protective mucosal immunity. A well-characterized linear B-cell epitope, D3(22-33) , from the fibronectin-binding protein A of Staphylococcus aureus was successfully introduced into PilVax and delivered intranasally to mice. Specific antipeptide immunoglobulin (Ig) G and IgA antibodies were detected in the serum and respiratory mucosa of vaccinated mice. Responses to the major pilus backbone protein Spy0128 were also assessed; robust antibody responses to this antigen were generated both systemically and in the respiratory and intestinal mucosa. Mice were challenged intranasally with the mouse-adapted S. aureus JSNZ strain and the S. aureus load quantified 7 days after challenge. Unexpectedly, exposure to PilVax, irrespective of the presence of the peptide, resulted in a significant reduction in S. aureus load in both the intestine and nasal mucosa (both P < 0.05) when compared with unvaccinated control mice. The mechanism(s) of protection are unclear, but merit further investigation to determine whether PilVax is a suitable platform for delivery of vaccine candidate antigens to the mucosa.