RESUMO
We have previously shown the ability of transamidated gluten (spf) to modulate both innate and adaptive intestinal immunity elicited by wheat gliadin in HLA-DQ8 transgenic mice (DQ8 mice), a model of gluten sensitivity. Herein, we evaluated the influence of spf when administered intragastrically on the immune response to native gliadin in DQ8 mice. To address the issue, we analysed three regimens of antigen administration: before immunisation (pre-treatment), during immunisation (co-treatment) and through breast milk during the lactating phase (suckling treatment). Mice were immunised mucosally by intranasal delivery of digested wheat gliadin along with cholera toxin in multiple doses. After sacrifice, isolated spleen and mesenteric lymph node (MLN) cells were challenged in vitro and the cytokine profile of culture supernatants assessed by ELISA and multiparametric assay. We found that only pre-treatment with spf was effective in down-regulating the gliadin-specific IFN-γ response and only in spleen cells. Interestingly, spf pre-treatment also induced systemic IL-6, IL-17A and TNF-α. By contrast, we found that spf pre-treatment upregulated INF-γ in MLN but also significantly decreased IL-2. In conclusion, our data provide evidence that the preventive intragastric administration of transamidated gluten is able to interfere with the classical cytokine profile induced by gliadin via mucosal immunisation in a transgenic model expressing one of the HLA molecules associated with coeliac disease.
Assuntos
Gliadina , Antígenos HLA-DQ , Camundongos Transgênicos , Triticum , Animais , Gliadina/imunologia , Antígenos HLA-DQ/imunologia , Camundongos , Triticum/imunologia , Feminino , Citocinas/metabolismo , Baço/imunologia , Doença Celíaca/imunologia , Humanos , Toxina da Cólera/farmacologia , Toxina da Cólera/imunologia , Toxina da Cólera/administração & dosagem , Interferon gama/metabolismo , Intestinos/imunologia , Linfonodos/imunologia , Linfonodos/efeitos dos fármacos , Imunização/métodos , Glutens/imunologia , Glutens/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-17/metabolismoRESUMO
Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection worldwide, potentially leading to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility if left untreated. Current strategies, including screening and antibiotics, have limited effectiveness due to high rates of asymptomatic cases and logistical challenges. A multiepitope prophylactic vaccine could afford long-term protection against infection. Immunoinformatic analyses were employed to design a multiepitope Chlamydia vaccine antigen. B- and T-cell epitopes from five highly conserved and immunogenic Ct antigens were predicted and selected for the vaccine design. The final construct, adjuvanted with cholera toxin A1 subunit (CTA1), was further screened for immunogenicity. CTA1-MECA (multiepitope Chlamydia trachomatis antigen) was identified as antigenic and nonallergenic. A tertiary structure was predicted, refined, and validated as a good quality model. Molecular docking exhibited strong interactions between the vaccine and toll-like receptor 4 (TLR4). Additionally, immune responses consistent with protection including IFN-γ, IgG + IgM antibodies, and T- and B-cell responses were predicted following vaccination in an immune simulation. Expression of the construct in an Escherichia coli expression vector proved efficient. To further validate the vaccine efficacy, we assessed its immunogenicity in mice. Immunization with CTA1-MECA elicited high levels of Chlamydia-specific antibodies in mucosal and systemic compartments.
Assuntos
Anticorpos Antibacterianos , Vacinas Bacterianas , Infecções por Chlamydia , Chlamydia trachomatis , Epitopos de Linfócito B , Epitopos de Linfócito T , Simulação de Acoplamento Molecular , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/genética , Infecções por Chlamydia/prevenção & controle , Infecções por Chlamydia/imunologia , Animais , Chlamydia trachomatis/imunologia , Epitopos de Linfócito T/imunologia , Camundongos , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Feminino , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Simulação por Computador , Epitopos/imunologia , Humanos , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Toxina da Cólera/imunologia , Toxina da Cólera/genética , Modelos Animais de DoençasRESUMO
Introduction: Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. Methods: We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. Results: CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Conclusion: Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.
Assuntos
Linfócitos T CD8-Positivos , Toxina da Cólera , Células Dendríticas , Melanoma Experimental , Camundongos Endogâmicos C57BL , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos , Linfócitos T CD8-Positivos/imunologia , Toxina da Cólera/imunologia , Toxina da Cólera/farmacologia , Melanoma Experimental/imunologia , Imunidade Inata , Feminino , Memória Imunológica , Imunidade TreinadaRESUMO
Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.
Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Genótipo , Vacinas contra Encefalite Japonesa , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/imunologia , Vírus da Encefalite Japonesa (Espécie)/classificação , Animais , Encefalite Japonesa/prevenção & controle , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Vacinas contra Encefalite Japonesa/imunologia , Vacinas contra Encefalite Japonesa/administração & dosagem , Vacinas contra Encefalite Japonesa/genética , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Humanos , Camundongos Endogâmicos BALB C , Feminino , Antígenos Virais/imunologia , Antígenos Virais/genética , Eficácia de Vacinas , Toxina da Cólera/genética , Toxina da Cólera/imunologiaRESUMO
Induction and regulation of specific intestinal immunoglobulin (Ig)A responses critically depend on dendritic cell (DC) subsets and the T cells they activate in the Peyer's patches (PP). We found that oral immunization with cholera toxin (CT) as an adjuvant resulted in migration-dependent changes in the composition and localization of PP DC subsets with increased numbers of cluster of differentiation (CD)103- conventional DC (cDC)2s and lysozyme-expressing DC (LysoDCs) in the subepithelial dome and of CD103+ cDC2s that expressed CD101 in the T cell zones, while oral ovalbumin (OVA) tolerization was instead associated with greater accumulation of cDC1s and peripherally induced regulatory T cells (pTregs) in this area. Decreased IgA responses were observed after CT-adjuvanted immunization in huCD207DTA mice lacking CD103+ cDC2s, while oral OVA tolerization was inefficient in cDC1-deficient Batf3-/- mice. Using OVA transgenic T cell receptor CD4 T cell adoptive transfer models, we found that co-transferred endogenous wildtype CD4 T cells can hinder the induction of OVA-specific IgA responses through secretion of interleukin-10. CT could overcome this blocking effect, apparently through a modulating effect on pTregs while promoting an expansion of follicular helper T cells. The data support a model where cDC1-induced pTreg normally suppresses PP responses for any given antigen and where CT's oral adjuvanticity effect is dependent on promoting follicular helper T cell responses through induction of CD103+ cDC2s.
Assuntos
Antígenos CD , Antígeno CD11b , Movimento Celular , Toxina da Cólera , Células Dendríticas , Tolerância Imunológica , Imunização , Imunoglobulina A , Cadeias alfa de Integrinas , Camundongos Knockout , Ovalbumina , Nódulos Linfáticos Agregados , Linfócitos T Reguladores , Animais , Camundongos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Cadeias alfa de Integrinas/metabolismo , Antígenos CD/metabolismo , Células Dendríticas/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Administração Oral , Toxina da Cólera/imunologia , Ovalbumina/imunologia , Ovalbumina/administração & dosagem , Linfócitos T Reguladores/imunologia , Antígeno CD11b/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Transferência Adotiva , Proteínas RepressorasRESUMO
A correlate of protection (CoP) is a measured adaptive immune response to vaccination or infection that is associated with protection against disease. However, the degree to which a CoP can serve as a surrogate end point for vaccine efficacy should depend on the robustness of this association. While cholera toxin is a dominant target of the human antibody response to Vibrio cholerae infection, antitoxin responses are not associated with long-term immunity, and are not effective CoPs for cholera. Instead, protection appears to be mediated by functional antibodies that target the O-polysaccharide coated V. cholerae outer membrane. Vibriocidal antibodies, which are complement-dependent bactericidal antibodies, remain the most accepted CoP for cholera and are used as surrogate end points in some vaccine studies. However, the association between vibriocidal antibody titers and immunity is not absolute, and they are unlikely to reflect a mechanistic correlate of protection against cholera.
Assuntos
Imunidade Adaptativa , Vacinas contra Cólera , Cólera/prevenção & controle , Eficácia de Vacinas , Vibrio cholerae/imunologia , Anticorpos Antibacterianos/imunologia , Toxina da Cólera/imunologia , Vacinas contra Cólera/efeitos adversos , Vacinas contra Cólera/imunologia , Humanos , Vibrio cholerae O1/imunologiaRESUMO
Human respiratory syncytial virus (hRSV) infection is a major pediatric health concern worldwide. Despite more than half a century of efforts, there is still no commercially available vaccine. In this study, we constructed and purified the recombinant protein CTA1-DD-RBF composed of a CTA1-DD mucosal adjuvant and prefusion F protein (RBF) using Escherichia coli BL21 cells. We studied the immunogenicity of CTA1-DD-RBF in mice. Intranasal immunization with CTA1-DD-RBF stimulated hRSV F-specific IgG1, IgG2a, sIgA, and neutralizing antibodies as well as T cell immunity without inducing lung immunopathology upon hRSV challenge. Moreover, the protective immunity of CTA1-DD-RBF was superior to that of the RBF protein, as confirmed by the assessment of serum-neutralizing activity and viral clearance after challenge. Compared to formalin-inactivated hRSV (FI-RSV), intranasal immunization with CTA1-DD-RBF induced a Th1 immune response. In summary, intranasal immunization with CTA1-DD-RBF is safe and effective in mice. Therefore, CTA1-DD-RBF represents a potential mucosal vaccine candidate for the prevention of human infection with hRSV.
Assuntos
Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/imunologia , Adjuvantes de Vacinas/administração & dosagem , Administração Intranasal , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Toxina da Cólera/genética , Feminino , Humanos , Imunidade nas Mucosas , Imunização , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Células Th1/imunologia , Proteínas Virais de Fusão/genética , Replicação ViralRESUMO
Vibrio cholerae is a bacterial pathogen which causes the severe acute diarrheal disease cholera. Given that a symptomatic incident of cholera can lead to long term protection, a thorough understanding of the immune response to this pathogen is needed to identify parameters critical to the generation and durability of immunity. To approach this, we utilized a live attenuated cholera vaccine to model the response to V. cholerae infection in 12 naïve subjects. We found that this live attenuated vaccine induced durable vibriocidal antibody titers that were maintained at least one year after vaccination. Similar to what we previously reported in infected patients from Bangladesh, we found that vaccination induced plasmablast responses were primarily specific to the two immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). Interestingly, the magnitude of the early plasmablast response at day 7 predicted the serological outcome of vaccination at day 30. However, this correlation was no longer present at later timepoints. The acute responses displayed preferential immunoglobulin isotype usage, with LPS specific cells being largely IgM or IgA producing, while cholera toxin responses were predominantly IgG. Finally, CCR9 was highly expressed on vaccine induced plasmablasts, especially on IgM and IgA producing cells, suggesting a role in migration to the gastrointestinal tract. Collectively, these findings demonstrate that the use of a live attenuated cholera vaccine is an effective tool to examine the primary and long-term immune response following V. cholerae exposure. Additionally, it provides insight into the phenotype and specificity of the cells which likely return to and mediate immunity at the intestinal mucosa. A thorough understanding of these properties both in peripheral blood and in the intestinal mucosae will inform future vaccine development against both cholera and other mucosal pathogens. Trial Registration: NCT03251495.
Assuntos
Anticorpos Antibacterianos/sangue , Vacinas contra Cólera/imunologia , Cólera/prevenção & controle , Imunidade Humoral , Vacinação , Vibrio cholerae O1 , Adulto , Linfócitos B/metabolismo , Toxina da Cólera/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G , Imunoglobulina M/metabolismo , Memória Imunológica , Lipopolissacarídeos/toxicidade , Masculino , Pessoa de Meia-Idade , Receptores CCR/genética , Receptores CCR/metabolismo , Fatores de Tempo , Vacinas Atenuadas/imunologia , Adulto JovemRESUMO
Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.
Assuntos
Doença Celíaca/tratamento farmacológico , Toxina da Cólera/administração & dosagem , Citocinas/metabolismo , Gliadina/administração & dosagem , Antígenos HLA-DQ/genética , Transglutaminases/metabolismo , Administração Intranasal , Animais , Doença Celíaca/genética , Doença Celíaca/imunologia , Toxina da Cólera/imunologia , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Regulação da Expressão Gênica , Gliadina/química , Gliadina/genética , Gliadina/imunologia , Antígenos HLA-DQ/metabolismo , Imunização , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologiaRESUMO
BACKGROUND: Cholera is an acute, diarrheal disease caused by Vibrio cholerae O1 or 139 that is associated with a high global burden. METHODS: We analyzed the estimated duration of immunity following cholera infection from available published studies. We searched PubMed and Web of Science for studies of the long-term immunity following cholera infection. We identified 22 eligible studies and categorized them as either observational, challenge, or serological. RESULTS: We found strong evidence of protection at 3 years after infection in observational and challenge studies. However, serological studies show that elevated humoral markers of potential correlates of protection returned to baseline within 1 year. Additionally, a subclinical cholera infection may confer lower protection than a clinical one, as suggested by 3 studies that found that, albeit with small sample sizes, most participants with a subclinical infection from an initial challenge with cholera had a symptomatic infection when rechallenged with a homologous biotype. CONCLUSIONS: This review underscores the need to elucidate potential differences in the protection provided by clinical and subclinical cholera infections. Further, more studies are warranted to bridge the gap between the correlates of protection and cholera immunity. Understanding the duration of natural immunity to cholera can help guide control strategies and policy.
Assuntos
Anticorpos Antibacterianos/sangue , Cólera/prevenção & controle , Memória Imunológica/imunologia , Vibrio cholerae O139/imunologia , Vibrio cholerae O1/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/imunologia , Criança , Pré-Escolar , Cólera/imunologia , Toxina da Cólera/imunologia , Proteção Cruzada/imunologia , Humanos , Lactente , Lipopolissacarídeos/imunologia , Pessoa de Meia-Idade , Adulto JovemRESUMO
The aim of this study was to assess the modulatory effect of TcpA in the expression of CEACAM1 adhesin molecule and IL-1, IL-8, and TNF-α pro-inflammatory cytokines in the Coculture model of Caco-2/PBMC (peripheral blood mononuclear cell) that can mimic the intestinal milieu. The TcpA gene from Vibrio cholerae ATCC14035 was cloned in pET-28a and transformed into Escherichia coli Bl-21. The recombinant TcpA-His6 protein was expressed and purified using Ni-column chromatography. The sequencing of transformed plasmid and Western blot analysis of purified protein confirmed the identity of rTcp. The cytotoxicity of different concentrations of recombinant protein for human colon carcinoma cell line (human colorectal adenocarcinoma cell [Caco-2 cell]) was assessed by MTT assay and showed viability of 92%, 82%, and 70%, for 10 µg/mL of TcpA after 24, 48, and 72 h, respectively. Co-cultures of Caco-2 and PBMCs were used to mimic the intestinal milieu and treated with different concentrations of rTcpA (1, 5, 10, and 50 µg/mL). Our data showed about 2.04-, 3.37-, 3.68-, and 42.7-fold increase in CEACAM1 gene expression, respectively, compared with the nontreated Caco-2/PBMC Coculture. Moreover, the expression of IL-1, IL-8, and TNF-α genes was significantly increased up to 15.75-, 7.04-, and 80.95-folds, respectively. In conclusion, V. cholerae TcpA induces statistically significant dose-dependent stimulatory effect on TNF-α, IL-,1, and IL-8 pro-inflammatory cytokines expression. Of these, TNF-α was much more affected which, consequently, elevated the CEACAM1 expression level in IECs. This suggests that TcpA protein is a critical effector as an inducer of increased adhesion potential of V. cholera as well as inflammatory responses of host intestinal tissue.
Assuntos
Toxina da Cólera/imunologia , Cólera , Proteínas de Fímbrias/imunologia , Leucócitos Mononucleares/imunologia , Vibrio cholerae , Antígenos CD/imunologia , Células CACO-2 , Moléculas de Adesão Celular/imunologia , Técnicas de Cocultura , Citocinas/imunologia , Humanos , Leucócitos Mononucleares/microbiologiaRESUMO
Deoxynivalenol (DON), a highly prevalent contaminant of grain-based products, is known to induce reproductive- and immunotoxicities. Considering the importance of immune development in early life, the present study investigated the effects of perinatal DON exposure on allergy development and vaccine responsiveness in the offspring. Pregnant mice received control or DON-contaminated diets (12.5 mg/kg diet) during pregnancy and lactation. After weaning, female offspring were sensitized to ovalbumin (OVA) by oral administration of OVA with cholera toxin (CT). Male offspring were injected with Influvac vaccine. OVA-specific acute allergic skin response (ASR) in females and vaccine-specific delayed-type hypersensitivity (DTH) in males were measured upon intradermal antigen challenge. Immune cell populations in spleen and antigen-specific plasma immunoglobulins were analyzed. In female CT+OVA-sensitized offspring of DON-exposed mothers ASR and OVA-specific plasma immunoglobulins were significantly higher, compared to the female offspring of control mothers. In vaccinated male offspring of DON-exposed mothers DTH and vaccine-specific antibody levels were significantly lower, compared to the male offspring of control mothers. In both models a significant reduction in regulatory T cells, Tbet+ Th1 cells and Th1-related cytokine production of the offspring of DON-exposed mothers was observed. In conclusion, early life dietary exposure to DON can adversely influence immune development in the offspring. Consequently, the immune system of the offspring may be skewed towards an imbalanced state, resulting in an increased allergic immune response to food allergens and a decreased immune response to vaccination against influenza virus in these models.
Assuntos
Hipersensibilidade Alimentar/imunologia , Imunogenicidade da Vacina , Vacinas contra Influenza/administração & dosagem , Lactação , Efeitos Tardios da Exposição Pré-Natal , Tricotecenos/toxicidade , Eficácia de Vacinas , Animais , Anticorpos Antivirais/sangue , Células Cultivadas , Toxina da Cólera/imunologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/metabolismo , Vacinas contra Influenza/imunologia , Masculino , Exposição Materna , Camundongos Endogâmicos C3H , Ovalbumina/imunologia , Gravidez , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , VacinaçãoRESUMO
This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4+ T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4+ T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.
Assuntos
Toxina da Cólera/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Lipossomos/imunologia , Pulmão/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteínas Recombinantes de Fusão/imunologia , Células Th1/imunologia , Células Th17/imunologia , Administração Intranasal , Animais , Apresentação de Antígeno , Células Cultivadas , Toxina da Cólera/metabolismo , Humanos , Imunogenicidade da Vacina , Imunoglobulina A/metabolismo , Vacinas contra Influenza/metabolismo , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/metabolismo , Peptídeos Cíclicos , Proteínas Recombinantes de Fusão/metabolismo , VacinaçãoRESUMO
Vibrio cholerae causes cholera and other infections, especially in children under five years of age. Cholera toxin (CT), toxin-coregulated pilus (TCP) and outer membrane protein W (OmpW) are three major virulence factors of this bacterium. The emergence of antimicrobial-resistant (AMR) strains and the absence of a comprehensive and flawless vaccine, has prompted other treatments. There are several advantages of egg yolk antibodies (IgY) over other immunotherapy agents, such as economic feasibility, high yield simple production, and better immune responsiveness to mammalian antigens due to phylogenetic distance. Accordingly, in the present study, IgYs against recombinant proteins CtxB (responsible for the CT binding to eukaryotic cells), TcpA (enhances bacterial attachment to enterocytes) and OmpW were produced, in single, coupled or combined forms, to evaluate and compare their protectivity potency. Immunoreactivity of IgYs were examined through protein and whole cell ELISA, their specificity was confirmed by western blotting, and their neutralizing effects on CT was evaluated in Y1 cell culture. Produced IgYs were gavage administered to different groups of infant mice infected with V. cholerae. The results indicated that IgYs produced against CtxB had the highest titers, and were able to neutralize cytotoxicity effects in Y1 cell culture, while the highest protection in the mice challenge was obtained by IgY-TcpA. No considerable increase was observed in immunoreactivity or protectivity of antibodies produced against combined antigens. The produced IgYs showed a good antigen-specificity and protectivity which can be used in passive immunotherapy against cholera.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Proteínas da Membrana Bacteriana Externa/imunologia , Toxina da Cólera/imunologia , Cólera/prevenção & controle , Gema de Ovo/imunologia , Proteínas de Fímbrias/imunologia , Imunoglobulinas/administração & dosagem , Vibrio cholerae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/efeitos adversos , Especificidade de Anticorpos , Linhagem Celular , Galinhas , Cólera/sangue , Cólera/imunologia , Cólera/microbiologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Imunização Passiva , Imunoglobulinas/imunologia , Camundongos Endogâmicos BALB C , Vibrio cholerae/patogenicidade , Fatores de VirulênciaRESUMO
BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored. RESULTS: Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer's patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals. CONCLUSIONS: The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Toxina da Cólera/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Lacticaseibacillus casei/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Administração Oral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Formação de Anticorpos/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Bovinos , Citocinas/imunologia , Trato Gastrointestinal/patologia , Trato Gastrointestinal/virologia , Imunidade Celular , Lacticaseibacillus casei/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Organismos Livres de Patógenos Específicos , Vacinas Sintéticas/imunologia , Carga ViralRESUMO
Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.
Assuntos
Adesinas de Escherichia coli/imunologia , Toxina da Cólera/imunologia , Infecções por Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Animais , Aotidae , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/prevenção & controle , Cobaias , Camundongos , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells (but not of CD4+CD25+FoxP3+ T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4+CD25+ T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.
Assuntos
Toxina da Cólera/imunologia , Fator IX/imunologia , Microbioma Gastrointestinal/imunologia , Hemofilia B/imunologia , Hemofilia B/microbiologia , Tolerância Imunológica/imunologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Células Vegetais/metabolismo , Administração Oral , Transferência Adotiva/métodos , Animais , Apresentação de Antígeno , Antígenos/genética , Antígenos/imunologia , Toxina da Cólera/genética , Modelos Animais de Doenças , Fator IX/genética , Deleção de Genes , Genoma de Cloroplastos , Lactuca/genética , Masculino , Camundongos , Camundongos Transgênicos , Plantas Geneticamente Modificadas , Linfócitos T Reguladores/imunologiaRESUMO
Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting.
Assuntos
Anticorpos Monoclonais/imunologia , Cólera/imunologia , Lipopolissacarídeos/imunologia , Vibrio cholerae O1/genética , Aglutinação , Animais , Anticorpos Antibacterianos/administração & dosagem , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Cólera/microbiologia , Toxina da Cólera/imunologia , Toxina da Cólera/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina G/imunologia , Lipídeo A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Transcriptoma , VirulênciaRESUMO
Influenza vaccines administered intramuscularly exhibit poor mucosal immune responses in the respiratory tract which is the prime site of the infection. Intranasal vaccination is a potential route for vaccine delivery which has been demonstrated effective in inducing protective immune responses in both systemic and mucosal compartments. For this purpose, nanoparticles have been used as antigen delivery systems to improve antigen capture by immune cells. In this paper we demonstrate efficient delivery of viral antigens to airway epithelial cells, macrophages and dendritic cells, using polysaccharide nanoparticles (NPL), leading to a strong protection against influenza virus infection. A formulation combining split Udorn virus antigens with NPL and the mucosal protein adjuvant CTA1-DD was administered intranasally and resulted in an enhanced specific humoral immune response. Furthermore, NPL carrying split Udorn, with or without CTA1-DD, inhibited virus transmission from infected to uninfected naive mice. These results demonstrate that an intranasal delivery system combining NPL, mucosal adjuvant CTA1-DD and split virus antigens confers robust protection against influenza infection and inhibits virus transmission.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos Virais/administração & dosagem , Toxina da Cólera/administração & dosagem , Portadores de Fármacos , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Nanopartículas , Infecções por Orthomyxoviridae/prevenção & controle , Polissacarídeos/química , Proteínas Recombinantes de Fusão/administração & dosagem , Adjuvantes Imunológicos/química , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/química , Antígenos Virais/imunologia , Toxina da Cólera/química , Toxina da Cólera/imunologia , Modelos Animais de Doenças , Composição de Medicamentos , Imunidade Humoral/efeitos dos fármacos , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Porosidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Nasal mucosal tissues are equipped with physical barriers, mucus and cilia, on their surface. The mucus layer captures inhaled materials, and the cilia remove the inhaled materials from the epithelial layer by asymmetrical beating. The effect of nasal physical barriers on the vaccine efficacy remains to be investigated. Tubulin tyrosine ligase-like family, member 1 (Ttll1) is an essential enzyme for appropriate movement of the cilia on respiratory epithelium, and its deficiency (Ttll1-KO) leads to mucus accumulation in the nasal cavity. Here, when mice were intra-nasally immunized with pneumococcal surface protein A (PspA, as vaccine antigen) together with cholera toxin (CT, as mucosal adjuvant), Ttll1-KO mice showed higher levels of PspA-specific IgA in the nasal wash and increased numbers of PspA-specific IgA-producing plasma cells in the nasal passages when compared with Ttll1 hetero (He) mice. Mucus removal by N-acetylcysteine did not affect the enhanced immune responses in Ttll1-KO mice versus Ttll1-He mice. Immunohistological and flow cytometry analyses revealed that retention time of PspA in the nasal cavity in Ttll1-KO mice was longer than that in Ttll1-He mice. Consistently, uptake of PspA by dendritic cells was higher in the nasopharynx-associated lymphoid tissue (NALT) of Ttll1-KO mice than that of Ttll1-He mice. These results indicate that the ciliary function of removing vaccine antigen from the NALT epithelial layer is a critical determinant of the efficacy of nasal vaccine.