RESUMO
Helicobacter pylori (H. pylori) is responsible for various chronic or acute diseases, such as stomach ulcers, dyspepsia, peptic ulcers, gastroesophageal reflux, gastritis, lymphoma, and stomach cancers. Although specific drugs are available to treat the bacterium's harmful effects, there is an urgent need to develop a preventive or therapeutic vaccine. Therefore, the current study aims to create a multi-epitope vaccine against H. pylori using lipid nanoparticles. Five epitopes from five target proteins of H. pylori, namely, Urease, CagA, HopE, SabA, and BabA, were used. Immunogenicity, MHC (Major Histocompatibility Complex) bonding, allergenicity, toxicity, physicochemical analysis, and global population coverage of the entire epitopes and final construct were carefully examined. The study involved using various bioinformatic web tools to accomplish the following tasks: modeling the three-dimensional structure of a set of epitopes and the final construct and docking them with Toll-Like Receptor 4 (TLR4). In the experimental phase, the final multi-epitope construct was synthesized using the solid phase method, and it was then enclosed in lipid nanoparticles. After synthesizing the construct, its loading, average size distribution, and nanoliposome shape were checked using Nanodrop at 280 nm, dynamic light scattering (DLS), and atomic force microscope (AFM). The designed vaccine has been confirmed to be non-toxic and anti-allergic. It can bind with different MHC alleles at a rate of 99.05%. The construct loading was determined to be about 91%, with an average size of 54 nm. Spherical shapes were also observed in the AFM images. Further laboratory tests are necessary to confirm the safety and immunogenicity of the multi-epitope vaccine.
Assuntos
Vacinas Bacterianas , Biologia Computacional , Helicobacter pylori , Nanopartículas , Helicobacter pylori/imunologia , Nanopartículas/química , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/química , Biologia Computacional/métodos , Humanos , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/química , Epitopos/imunologia , Epitopos/química , Simulação de Acoplamento Molecular , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/química , Infecções por Helicobacter/prevenção & controle , Infecções por Helicobacter/imunologia , Receptor 4 Toll-Like/imunologia , Urease/imunologia , Urease/química , Imunoinformática , LipossomosRESUMO
Helicobacter pylori causes globally prevalent infections that are highly related to chronic gastritis and even development of gastric carcinomas. With the increase of antibiotic resistance, scientists have begun to search for better vaccine design strategies to eradicate H. pylori colonization. However, while current strategies prefer to formulate vaccines with a single H. pylori antigen, their potential has not yet been fully realized. Outer membrane vesicles (OMVs) are a potential platform since they could deliver multiple antigens. In this study, we engineered three crucial H. pylori antigen proteins (UreB, CagA, and VacA) onto the surface of OMVs derived from Salmonella enterica serovar Typhimurium (S. Typhimurium) mutant strains using the hemoglobin protease (Hbp) autotransporter system. In various knockout strategies, we found that OMVs isolated from the ΔrfbP ΔfliC ΔfljB ΔompA mutants could cause distinct increases in immunoglobulin G (IgG) and A (IgA) levels and effectively trigger T helper 1- and 17-biased cellular immune responses, which perform a vital role in protecting against H. pylori. Next, OMVs derived from ΔrfbP ΔfliC ΔfljB ΔompA mutants were used as a vector to deliver different combinations of H. pylori antigens. The antibody and cytokine levels and challenge experiments in mice model indicated that co-delivering UreB and CagA could protect against H. pylori and antigen-specific T cell responses. In summary, OMVs derived from the S. Typhimurium ΔrfbP ΔfliC ΔfljB ΔompA mutant strain as the vector while importing H. pylori UreB and CagA as antigenic proteins using the Hbp autotransporter system would greatly benefit controlling H. pylori infection.
Outer membrane vesicles (OMVs), as a novel antigen delivery platform, has been used in vaccine design for various pathogens and even tumors. Salmonella enterica serovar Typhimurium (S. Typhimurium), as a bacterium that is easy to engineer and has both adjuvant efficacy and immune stimulation capacity, has become the preferred bacterial vector for purifying OMVs after Escherichia coli. This study focuses on the design of Helicobacter pylori ;(H. pylori) vaccines, utilizing genetically modified Salmonella OMVs to present several major antigens of H. pylori, including UreB, VacA and CagA. The optimal Salmonella OMV delivery vector and antigen combinations are screened and identified, providing new ideas for the development of H. pylori vaccines and an integrated antigen delivery platform for other difficult to develop vaccines for bacteria, viruses, and even tumors.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Infecções por Helicobacter , Helicobacter pylori , Salmonella typhimurium , Animais , Infecções por Helicobacter/prevenção & controle , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Helicobacter pylori/imunologia , Helicobacter pylori/genética , Camundongos , Salmonella typhimurium/imunologia , Salmonella typhimurium/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/genética , Feminino , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Imunoglobulina G , Engenharia Genética , Urease/imunologia , Urease/genética , Modelos Animais de DoençasRESUMO
As a key virulence factor for persistent colonization, urease B subunit (UreB) is considered to be an ideal vaccine antigen against Helicobacter pylori infection. However, the role and molecular mechanisms of UreB involved in immune microenvironment dysregulation still remain largely unknown. In the present study, we evaluated the effects of UreB on macrophage activation and found that UreB induced PD-L1 accumulation on bone marrow-derived macrophages (BMDMs). Co-culture assays further revealed that UreB-induced PD-L1 expression on BMDMs significantly decreased the proliferation and secretion of cytolytic molecules (granzyme B and perforin) of splenic CD8+ T cells isolated from inactivated H. pylori-immunized mice. More importantly, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation techniques, it has been confirmed that myosin heavy chain 9 (Myh9) is a direct membrane receptor for UreB and is required for PD-L1 up-regulation on BMDMs. Molecular studies further demonstrated that the interaction between UreB and Myh9 decreased GCN2 autophosphorylation and enhanced the intracellular pool of amino acids, leading to the up-regulation of S6K phosphorylation, a commonly used marker for monitoring activation of mTORC1 signaling activity. Furthermore, blocking mTORC1 activation with its inhibitor Temsirolimus reversed the UreB-induced PD-L1 up-regulation and the subsequent inhibitory effects of BMDMs on activation of cytotoxic CD8+ T-cell responses. Overall, our data unveil a novel immunosuppressive mechanism of UreB during H. pylori infection, which may provide valuable clues for the optimization of H. pylori vaccine.
Assuntos
Antígeno B7-H1/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Cadeias Pesadas de Miosina/imunologia , Urease/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Células THP-1RESUMO
BACKGROUND: Helicobacter pylori is a gram-negative bacterium involved in many gastric pathologies such as ulcers and cancers. Although the treatment for this infection has existed for several years, the development of a vaccine is nevertheless necessary to reduce the severe forms of the disease. For more than three decades, many advances have been made particularly in the understanding of virulence factors as well as the pathogenesis of gastric diseases caused by H. pylori. Among these key virulence factors, specific antigens have been identified: Urease, Vacuolating cytotoxin A (VacA), Cytotoxin-associated gene A (CagA), Blood group antigen-binding adhesin (BabA), H. pylori adhesin A (HpaA), and others. OBJECTIVES: This review will focus on H. pylori adhesins, in particular, on HpaA and on the current knowledge of H. pylori vaccines. METHODS: All of the information included in this review was retrieved from published studies on H. pylori adhesins in H. pylori infections. RESULTS: These proteins, used in their native or recombinant forms, induce protection against H. pylori in experimental animal models. CONCLUSION: H. pylori adhesins are known to be promising candidate vaccines against H. pylori. Future research should be carried out on adhesins, in particular, on HpaA.
Assuntos
Adesinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Helicobacter , Helicobacter pylori , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Urease/imunologia , Fatores de Virulência/imunologiaRESUMO
Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.
Assuntos
Vacinas Bacterianas/administração & dosagem , Infecções por Helicobacter , Imunogenicidade da Vacina , Adjuvantes Imunológicos , Administração Oral , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Imunização/métodos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Urease/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Urease is an effective target for design of a therapeutic epitope vaccine against Helicobacter pylori (H. pylori). In our previous studies, an epitope vaccine CTB-UE containing Th and B epitopes from H. pylori urease was constructed, and the CTB-UE vaccine could provide therapeutic effect on H. pylori infection in mice. However, a multivalent vaccine, combining different antigens participating in different aspects of H. pylori colonization and pathogenesis, may be more effective as a therapeutic vaccine than a univalent vaccine targetting urease. Therefore, a multivalent epitope vaccine FVpE, containing Th1-type immune adjuvant NAP, three selected functional fragments from CagA and VacA, and an urease multi-epitope peptide (UE) from CTB-UE, was constructed in this study and expected to obtain better sterilizing immunity than the univalent epitope vaccine CTB-UE. The therapeutic effect of multivalent epitope vaccine FVpE with polysaccharide adjuvant (PA) was evaluated in H. pylori-infected Mongolian gerbil model. The results showed that both FvpE and CTB-UE vaccine could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect on H. pylori urease activity. However, only FVpE could induce high levels of specific antibodies to CagA, VacA, and NAP. In addition, oral therapeutic immunization with FVpE plus PA significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils compared with oral immunization with CTB-UE plus PA, or FVpE only, and the FVpE vaccine with PA even exhibited sterilizing immunity. The protection of FVpE was related to the mixed CD4+ T cell responses and epitope-specific antibodies against various H. pylori antigens. These results indicate that a multivalent epitope vaccine targetting various H. pylori antigens could be a promising candidate against H. pylori infection.
Assuntos
Vacinas Bacterianas/administração & dosagem , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Masculino , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Polissacarídeos/imunologia , Estômago/efeitos dos fármacos , Estômago/imunologia , Estômago/microbiologia , Urease/imunologia , Vacinação/métodosRESUMO
Helicobacter pylori (H. pylori) urease is a key protein for persistent infection of the bacteria in the stomach. Although H. pylori generally induce anti-H. pylori-specific antibodies (Abs), these Abs do not usually work for eradication or prevention of the H. pylori infection. In our previous study, we identified a linear epitope composed of 19-mer peptides termed UB-33, CHHLDKSIKEDVQFADSRI, within the large subunit of H. pylori urease. Anti-UB-33-specific Abs neutralized the enzymatic activity of H. pylori urease in vitro. In the present study, we evaluated the effect of immunization of BALB/c mice with H. pylori UB-33 peptide. After confirming the production of anti-UB-33-specific Abs, mice were challenged orally with H. pylori Sydney Strain-1 (SS-1). Mice producing anti-UB-33-specific Abs were not infected with SS-1, and the amount of SS-1 isolate in their stomach was significantly reduced. Also, the urease-negative mutant of H. pylori, HPP1801, did not colonize in the stomach, indicating that H. pylori urease was a critical element for infection of H. pylori in the gastric mucosa. Moreover, mice producing UB-33-specific Abs apparently suppressed H. pylori infection in the stomach where anti-UB-33 Abs were secreted in the gastric juice, indicating that H. pylori colonization was inhibited in the presence of anti-UB-33 Abs. In addition, the neutralization activity of sera from mice immunized with purified urease was less potent than that in the sera from mice immunized with UB-33. Furthermore, the recognition of epitope UB-33 was mediated through Toll-like receptor 2 (TLR2) on the B-1 cells using TLR2-knockout BALB/c mice in vivo. These results indicate that liner peptide UB-33 should be used for immunization to induce neutralizing Abs instead of purified H. pylori urease to prevent H. pylori infection and their colonization in the stomach.
Assuntos
Anticorpos Antibacterianos/biossíntese , Anticorpos Neutralizantes/biossíntese , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/prevenção & controle , Imunização/métodos , Peptídeos/imunologia , Urease/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Epitopos/química , Epitopos/imunologia , Feminino , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/microbiologia , Deleção de Genes , Expressão Gênica , Infecções por Helicobacter/genética , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/imunologia , Soros Imunes/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/administração & dosagem , Peptídeos/síntese química , Estômago/efeitos dos fármacos , Estômago/imunologia , Estômago/microbiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Urease/deficiência , Urease/genéticaRESUMO
Helicobacter pylori is a pathogenic bacterium that infects the stomach, causing chronic gastritis; and it is also considered to be related to the occurrence of gastric cancers. Although some eradication regimens including multiple antibiotics have been developed, the emergence of resistance to antibiotics becomes problematic. Therefore, other approaches to compensate or augment the effects of standard regimens are needed. In this study, we examined the possible synergistic effects of anti-H. pylori urease IgY and Lactobacillus johnsonii No.1088 (LJ88) both in vitro and in vivo. Anti-H. pylori urease IgY was purified from egg yolks laid by the hens immunized with urease purified from H. pylori. LJ88 is a unique strain of lactic acid bacterium isolated from human gastric juice, and it has been reported to inhibit H. pylori both in vitro and in vivo. The in vitro mixed culture study showed that anti-H. pylori urease IgY augmented the anti-H. pylori activity of LJ88 against both clarithromycin-sensitive and -resistant H. pylori strains. In a germ-free mice infection model, combined administration of daily anti-H. pylori urease IgY and weekly living LJ88 significantly reduced H. pylori infections, whereas either monotherapy did not. In an in vivo human gut microbiota-associated mice model, not only daily administration of living LJ88 but also heat-killed one significantly reduced an H. pylori infection in the stomach when combined with anti-H. pylori urease IgY. The extent of reduction of the stomach H. pylori by such a combination therapy was larger than that reported for LJ88 monotherapy. These results taken together revealed a synergistic effect of anti-H. pylori urease IgY and living or heat-killed LJ88, thus suggesting that such a combination might be a promising therapy to possibly compensate and/or augment standard anti-H. pylori regimens.
Assuntos
Anticorpos Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Imunoglobulinas/farmacologia , Lactobacillus johnsonii/fisiologia , Probióticos/farmacologia , Urease/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Galinhas/imunologia , Gema de Ovo/imunologia , Feminino , Vida Livre de Germes , Infecções por Helicobacter/prevenção & controle , Infecções por Helicobacter/terapia , Humanos , Imunização , Imunoglobulinas/imunologia , Camundongos , Microbiota , Organismos Livres de Patógenos Específicos , Estômago/imunologia , Estômago/microbiologia , Urease/farmacologiaRESUMO
Helicobacter pylori bacteria are involved in gastroduodenal disorders, including gastric adenocarcinoma. Since the current therapies encounter with some significant shortcomings, much attention has been paid to the development of new alternative diagnostic and treatment modalities such as immunomedicines to target H. pylori. Having used phage display technology, we isolated fully humane small antibody (Ab) fragment (VL) against the Flap region of urease enzyme of H. pylori to suppress its enzymatic activity. Solution biopanning (SPB) and screening process against a customized biotinylated peptide corresponding to the enzyme Flap region resulted in the selection of VL single domain Abs confirmed by the enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. The selected Ab fragments showed a high affinity with a KD value of 97.8 × 10-9 and specificity to the enzyme with high inhibitory impact. For the first time, a VL single domain Ab was isolated by SPB process against a critical segment of H. pylori urease using a diverse semi-synthetic library. Based on our findings, the selected VL Ab fragments can be used for the diagnosis, imaging, targeting, and/or immunotherapy of H. pylori. Further, Flap region shows great potential for vaccine therapy.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Helicobacter pylori/enzimologia , Anticorpos de Domínio Único/imunologia , Urease/imunologia , Afinidade de Anticorpos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Técnicas de Visualização da Superfície Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/imunologia , Humanos , Biblioteca de Peptídeos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Urease/antagonistas & inibidores , Urease/químicaRESUMO
Some plant polysaccharides (PPSs) had been used as the adjuvants for systemic vaccination. In this study, we investigated whether PPSs could exhibit adjuvant effect at the mucosa. Groups of mice were intranasally immunized with Epimedium Polysaccharide (EPS), Trollius chinensis polysaccharide (TCPS), Siberian solomonseal rhizome polysaccharide (SSRPS) and Astragalus polysaccharides (APS) together with ovalbumin (OVA). Significantly higher levels of OVA-specific IgG in serum and secretory IgA in saliva, vaginal wash and intestinal lavage fluid were induced after immunization with OVA plus one of the four PPSs compared to OVA alone. Antigen absorption and TLR2 (Toll-like receptor 2) activation may be related to their mucosal adjuvant effect. Of note, when APS used as an adjuvant, intranasally vaccination with recombination UreB (rUreB, Urease subunit B) conferred more robust protection against Helicobacter pylori (H. pylori). Immunized with rUreB in combination APS resulted in mixed specific Th1 and Th17 immune response, which may contribute to the inhibition of H. pylori colonization. Though specific Th2-dominant responses were elicited when the other three PPS intranasally immunized with rUreB, no significant difference in the protective effect were found between those groups and rUreb alone group. Taken together, the four PPSs may be promising candidates for mucosal adjuvant, and APS could enhance rUreB-specific protective immunity against H. pylori infection.
Assuntos
Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Mucosa/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal/métodos , Animais , Feminino , Imunização/métodos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/microbiologia , Polissacarídeos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Urease/imunologia , Vacinação/métodosRESUMO
Helicobacter pylori (H. pylori) as microaerophilic, Gram-negative bacterium colonize the human gastric milieu, where it impetuses chronic disorders. Vaccination is a complementary plan, along with antibiotic therapy, for clearance of H. pylori. Today, Computer based tools are essential for the evaluation, design, and experiment for novel chimeric targets for immunological administration. The purpose of this experiment was immunoinformatic analysis of UreB and HpaA molecules in a fusion arrangement and also, construction and expression of recombinant protein containing chimeric sequences. The targets sequences were screened by using of standard in silico tools and immunoinformatic web servers. The high-resolution 3D models of the protein were created and were validated; indeed, the B-and T-cell restricted epitopes were mapped on the chimeric protein. The recombinant protein in frame of the expression vector pET28a were expressed and purified successfully. The urease activity and immunoblotting were performed in vitro condition. This study confirmed that the engineered protein as a highly conserved, hydrophilic, non-allergenic contained remarkable B-cell and T-cell epitopes. It was magnificently attained; chimeric UreB229-561-HpaA could provoke both humoral and cellular immunity. The immunoblotting was shown that the chimeric protein could be detected by serum of immunized animal and H.pylori positive patients. In this study, several antigenic patches from UreB and HpaA were identified that could be an efficient immune system activator. The in vitro analysis of our chimeric molecule confirmed its urease activity. It also confirmed that the chimeric protein could be detected by serum of immunized animal and H.pylori positive patients.
Assuntos
Adesinas Bacterianas/química , Proteínas Recombinantes de Fusão/química , Urease/química , Adesinas Bacterianas/imunologia , Sequência de Aminoácidos , Animais , Simulação por Computador , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Meia-Vida , Helicobacter pylori/imunologia , Humanos , Modelos Moleculares , Conformação Proteica em Folha beta , Engenharia de Proteínas , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Urease/imunologiaRESUMO
Vaccination is an increasingly important alternative approach to control Helicobacter pylori infection, since H. pylori resistance to previously efficacious antibiotic regimens is increased, and H. pylori eradication treatment for upper gastrointestinal diseases is becoming less successful. Fortunately, an efficient oral monovalent H. pylori vaccine has been developed. However, compared with monovalent vaccines, multivalent vaccines have the potential to induce more effective and comprehensive protection against H. pylori infection. In this study, we designed and produced a multivalent epitope-based vaccine cholera toxin B subunit (CTB)-HUUC with the intramucosal adjuvant CTB and tandem copies of B-cell epitopes (HpaA132-141, UreA183-203, and UreB321-339) and T-cell epitopes (HpaA88-100, UreA27-53, UreB229-251, UreB317-329, UreB373-385, UreB438-452, UreB546-561, CagA149-164, and CagA196-217) from H. pylori adhesion A subunit (HpaA), urease A subunit (UreA), urease B subunit (UreB), and cytotoxin-associated antigen (CagA). Serum IgG, stomach, and intestine mucosal sIgA from mice after CTB-HUUC vaccination neutralized H. pylori urease activity in vitro. CTB-HUUC vaccination promoted H. pylori-specific lymphocyte responses and a mixed CD4+ T cell immune response as indicated by IFN-γ, interleukin-4, and interleukin-17 production in mice. Both oral prophylactic and therapeutic CTB-HUUC vaccinations reduced gastric urease activity and H. pylori infection and protected stomachs in mice. Taken together, CTB-HUUC is a promising potent and safe multivalent vaccine in controlling H. pylori infection in BALB/c mouse model.
Assuntos
Toxina da Cólera/imunologia , Vacinas contra Cólera/imunologia , Infecções por Helicobacter/prevenção & controle , Urease/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Toxina da Cólera/administração & dosagem , Vacinas contra Cólera/administração & dosagem , Modelos Animais de Doenças , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Urease/administração & dosagem , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Brucella (B) species are brucellosis causative agents, a worldwide zoonotic illness causing Malta fever in humans and abortion in domestic animals. In this work, we evaluated the vaccine potential of Trimethyl chitosan (TMC) nanoparticles formulation of Urease (TMC/Urease) against brucellosis. TMC/Urease nanoparticles and urease without any adjuvant were separately administered both orally and intraperitoneally. Intraperitoneal (i.p.) administration of urease alone as well as oral administration of both TMC/Urease nanoparticles and urease alone, elicited low titers of specific immunoglobulin G (IgG), while i.p. immunization with TMC/Urease nanoparticles induced high specific IgG production levels. As it was indicated by the cytokine assay and the antibody isotypes, i.p. immunization by urease alone, and TMC/Urease nanoparticles induced a mixed Th1-Th2 immune response, whereas oral administration of both urease alone and TMC/Urease nanoparticles induced a mixed Th1-Th17 immune response. In lymphocyte proliferation assay, spleen cells from i.p.-vaccinated mice with TMC/Urease nanoparticles showed a strong recall proliferative response. Vaccinated animals were challenged with virulent strains of B. melitensis and B. abortus. I.p. vaccination with TMC/Urease nanoparticles resulted in a high degree of protection. Altogether, our results indicated that TMC nanoparticles are a potent delivery system for i.p.-administered Brucella antigens.
Assuntos
Vacina contra Brucelose/imunologia , Brucelose , Quitosana/uso terapêutico , Nanopartículas/uso terapêutico , Urease/imunologia , Urease/uso terapêutico , Animais , Brucella abortus/imunologia , Brucella melitensis/imunologia , Brucelose/tratamento farmacológico , Brucelose/imunologia , Brucelose/prevenção & controle , Humanos , Imunoglobulina G/metabolismo , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , VacinaçãoRESUMO
BACKGROUND: Gram-positive enhancer matrix particles (GEM) produced by Lactococcus lactis can enhance vaccine-induced immune response. However, the mechanism under which this adjuvant mounts the efficacy of orally administered vaccines remains unexplored. MATERIALS AND METHODS: We used a prophylactic mice model to investigate the mechanism of GEM-adjuvanted vaccination. Helicobacter pylori urease-specific antibody response was monitored and detected in murine serum by ELISA. Urease-specific splenic cytokine profile was examined. Gastric inflammatory responses were measured on day 43 or 71 by quantitative real-time PCR, flow cytometry and histology. RESULTS: We found that GEM enhanced the efficiency of oral H. pylori vaccine by promoting innate immunity. The vaccine CUE-GEM composed of GEM particles and recombinant antigen CTB-UE provided protection of immunized mice against H. pylori insult. The protective response was associated with induction of postimmunization gastritis and local Th1/Th17 cell-medicated immune response. We showed that innate inflammatory responses including neutrophil chemokines CXCL1-2, neutrophils, and antimicrobial proteins S100A8 and MUC1 were significantly elevated. Within all infected mice, S100A8 and MUC1 levels were negatively correlated with H. pylori burden. Strikingly, mice receiving GEM also show reduction of colonization, possibly through natural host response pathways to recruit CD4+ T cells and promote S100A8 expression. CONCLUSIONS: These findings suggest that GEM-based vaccine may impact Th1/Th17 immunity to orchestrate innate immune response against H. pylori infection.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Bacterianas/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Imunidade Inata , Lactococcus lactis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/isolamento & purificação , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Mucosa Gástrica/patologia , Infecções por Helicobacter/patologia , Leucócitos Mononucleares/imunologia , Masculino , Camundongos Endogâmicos BALB C , Urease/imunologiaRESUMO
Epitope-based vaccine is a promising strategy for therapeutic vaccination against Helicobacter pylori (H. pylori) infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori, remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP), heat shock protein 60 (HSP60) and H. pylori adhesin A (HpaA) was constructed based on mucosal adjuvant cholera toxin B subunit (CTB), Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes (UreA27-53, UreA183-203, HpaA132-141, and HSP60189-203), and also the epitope-rich regions of urease B subunit (UreB158-251 and UreB321-385) predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori-infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes (UreB158-172, UreB181-195, UreB211-225, UreB349-363, HpaA132-141, and HSP60189-203). In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4+ T cell (Th cell) response, IgG, and secretory IgA (sIgA) antibodies to H. pylori. These results indic ate that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.
Assuntos
Adesinas Bacterianas/imunologia , Chaperonina 60/imunologia , Epitopos/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Imunização , Urease/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Linfócitos T CD4-Positivos , Toxina da Cólera/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Gerbillinae , Infecções por Helicobacter/imunologia , Helicobacter pylori/genética , Imunoglobulina A Secretora , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Estômago/microbiologia , Estômago/patologia , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and adhesins, which are on the surface of H. pylori, play a pivotal role in binding to human gastric mucosa. MATERIALS AND METHODS: In the present study, we constructed a multivalent epitope-based vaccine named CFAdE with seven carefully selected antigenic fragments from four H. pylori adhesins (urease, Lpp20, HpaA and CagL). The specificity, immunogenicity and ability to produce neutralizing antibodies of CFAdE were evaluated in BALB/c mice. After that, its therapeutic efficacy and protective immune mechanisms were explored in H. pylori-infected Mongolian gerbils. RESULTS: The results indicated that CFAdE could induce comparatively high levels of specific antibodies against urease, Lpp20, HpaA and CagL. Additionally, oral therapeutic immunization with CFAdE plus polysaccharide adjuvant (PA) significantly decreased H. pylori colonization compared with oral immunization with urease plus PA, and the protection was correlated with IgG and sIgA antibody and antigen-specific CD4+ T cells. CONCLUSIONS: This study indicated that the multivalent epitope-based vaccine, which targeted multiple adhesins in adherence of H. pylori to the gastric mucosa, is more effective than the univalent vaccine targeting urease only. This multivalent epitope-based vaccine may be a promising therapeutic candidate vaccine against H. pylori infection.
Assuntos
Adesinas Bacterianas/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções por Helicobacter/terapia , Helicobacter pylori/imunologia , Lipoproteínas/imunologia , Urease/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Vacinas Bacterianas/administração & dosagem , Modelos Animais de Doenças , Epitopos/imunologia , Gerbillinae , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Camundongos Endogâmicos BALB C , Resultado do TratamentoRESUMO
To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease ß subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/análise , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/imunologia , Hidroliases/análise , Oxirredutases/análise , Fatores de Alongamento de Peptídeos/análise , Piruvato Sintase/análise , Urease/análise , Proteínas de Bactérias/imunologia , Biomarcadores/análise , Feminino , Humanos , Hidroliases/imunologia , Immunoblotting , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Masculino , Oxirredutases/imunologia , Fatores de Alongamento de Peptídeos/imunologia , Mapeamento de Peptídeos , Piruvato Sintase/imunologia , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Urease/imunologiaRESUMO
Helicobacter pylori have colonized the gastric mucosa of half of the population worldwide. This bacterium is classified as a definitive type I carcinogen by the World Health Organization and no effective vaccine has been found against it yet. Thus, a logical and rational vaccine design against H. pylori is necessary. Because of its tremendous complexity and elicited immune responses, the vaccine design should considered multiple antigens to enhance immune-protection, involved in the different stages of pathogenesis besides inducing a specific immune response by B- and T-cell multi-epitopes. In this study, emphasis was placed on the design of a new unique vaccine named CTB-multiHp. In silico techniques were used to design a chimeric construct consisting of cholera toxin B subunit fused to multi-epitope of urease B (residue 148-158, 188-198), cytotoxin-associated gene A (residue 584-602), neutrophil activating protein (residue 4-28), vacuolating cytotoxin gene A (residue 63-81), H. pylori adhesine A (residue77-99), heat shock protein A (residue 32-54) and gamma glutamyl transpeptidase (residue 271-293). The tertiary structure and features of the vaccine were analyzed. The chimeric protein was expressed in Escherichia coli BL21 and the serology analyses indicated that the CTB-multiHp protein produced exhibit immune-reactivity. The results showed that CTB-multiHp could be a good vaccine candidate against H. pylori. Ongoing studies will evaluate the effects of CTB-multiHp against H. pylori infection.
Assuntos
Vacinas Bacterianas/imunologia , Epitopos/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Proteínas Recombinantes de Fusão/química , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/genética , Toxina da Cólera/química , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Clonagem Molecular , Desenho de Fármacos , Epitopos/química , Epitopos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Humanos , Interleucina-8/química , Interleucina-8/genética , Interleucina-8/imunologia , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Urease/química , Urease/genética , Urease/imunologia , gama-Glutamiltransferase/química , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/imunologiaRESUMO
BACKGROUND/AIMS: The Helicobacter pylori prevalence has been decreasing in many parts of the world. This study aimed to investigate the current epidemiological status of H. pylori infection in urban China. MATERIALS AND METHODS: The study included 51,299 subjects aged ≥18 years who underwent health checkups between April 2013 and June 2016 in a city of China. H. pylori infection was determined by detecting H. pylori urease-IgG antibodies. Statistical analyses included chi-square tests for trends and curve fitting. RESULTS: The overall H. pylori prevalence was found to be 31.9%, with the highest prevalence in the 1950-1959 birth cohort. It was lower in the subsequent birth cohorts (trends, p<0.001). The decrease in H. pylori prevalence was correlated with the increase in per capita gross domestic product (GDP) and real per capita GDP; the power model was best fitted (R2=0.914 and 0.997 and p=0.011 and 0.000, respectively). CONCLUSION: There has been a striking decrease in the H. pylori prevalence in urban China. The birth cohort effect and economic growth are the most likely causes of this phenomenon.
Assuntos
Infecções por Helicobacter/epidemiologia , Helicobacter pylori , População Urbana/estatística & dados numéricos , Anticorpos Antibacterianos/análise , Distribuição de Qui-Quadrado , China/epidemiologia , Estudos de Coortes , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Prevalência , Urease/imunologiaRESUMO
Despite the recent description of the mucosal vaccine-induced reduction of Helicobacter pylori natural infection in a phase 3 clinical trial, the absence of immune correlates of protection slows the final development of the vaccine. In this study, we evaluated the role of interleukin (IL)-22 in mucosal vaccine-induced protection. Gastric IL-22 levels were increased in mice intranasally immunized with urease+cholera toxin and challenged with H. felis, as compared with controls. Flow cytometry analysis showed that a peak of CD4+IL-22+IL-17+ T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice. The inhibition of the IL-22 biological activity prevented the vaccine-induced reduction of H. pylori infection. Remarkably, anti-microbial peptides (AMPs) extracted from the stomachs of vaccinated mice, but not from the stomachs of non-immunized or immunized mice, injected with anti-IL-22 antibodies efficiently killed H. pylori in vitro. Finally, H. pylori infection in vaccinated RegIIIß-deficient mice was not reduced as efficiently as in wild-type mice. These results demonstrate that IL-22 has a critical role in vaccine-induced protection, by promoting the expression of AMPs, such as RegIIIß, capable of killing Helicobacter. Therefore, it can be concluded that urease-specific memory Th17/Th22 cells could constitute immune correlates of vaccine protection in humans.