RESUMO
The contribution of Toll-like receptor (TLR) signaling to T cell-dependent (TD) antibody responses was assessed by using mice lacking the TLR signaling adaptor MyD88 in individual cell types. When a soluble TLR9 ligand was used as adjuvant for a protein antigen, MyD88 was required in dendritic cells but not in B cells to enhance the TD antibody response, regardless of the inherent immunogenicity of the antigen. In contrast, a TLR9 ligand contained within a virus-like particle substantially augmented the TD germinal center IgG antibody response, and this augmentation required B cell MyD88. The ability of B cells to discriminate between antigens based on the physical form of a TLR ligand probably reflects an adaptation to facilitate strong antiviral antibody responses.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais , Receptor Toll-Like 9/imunologia , Animais , Anticorpos Antivirais/sangue , Células Dendríticas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Imunológicos , Orthomyxoviridae/imunologiaRESUMO
BACKGROUND: Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic. METHODS: A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qß phage particle (Qß-CSP). RESULTS: Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qß-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qß-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity. CONCLUSIONS: Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.
Assuntos
Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Feminino , Imunogenicidade da Vacina , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologiaRESUMO
Interleukin-1ß (IL-1ß) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1ß activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1ß. The vaccine hIL1bQb consisting of full-length, recombinant IL-1ß coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1ß-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1ß-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1ß and represents a new treatment option for IL-1ß-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Diabetes Mellitus Tipo 2/terapia , Interleucina-1beta/imunologia , Vacinas/administração & dosagem , Adulto , Idoso , Animais , Diabetes Mellitus Tipo 2/imunologia , Método Duplo-Cego , Feminino , Humanos , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Vacinas/imunologiaRESUMO
Extensive studies have been undertaken to describe naive B cells differentiating into memory B cells at a cellular and molecular level. However, relatively little is known about the fate of memory B cells upon Ag re-encounter. We have previously established a system based on virus-like particles (VLPs), which allows tracking of VLP-specific B cells by flow cytometry as well as histology. Using allotype markers, it is possible to adoptively transfer memory B cells into a naive mouse and track responses of naive and memory B cells in the same mouse under physiological conditions. We have observed that VLP-specific memory B cells quickly differentiated into plasma cells that drove the early onset of a strong humoral IgG response. However, neither IgM(+) nor IgG(+) memory B cells proliferated extensively or entered germinal centers. Remarkably, plasma cells derived from memory B cells preferentially homed to the bone marrow earlier and secreted increased levels of Abs when compared with primary plasma cells derived from naive B cells. Hence, memory B cells have the unique phenotype to differentiate into highly effective secondary plasma cells.
Assuntos
Allolevivirus/imunologia , Anticorpos Antivirais/imunologia , Diferenciação Celular/imunologia , Memória Imunológica , Plasmócitos/imunologia , Animais , Imunização , CamundongosRESUMO
The lung is an important entry site for pathogens; its exposure to antigens results in systemic as well as local IgA and IgG antibodies. Here we show that intranasal administration of virus-like particles (VLPs) results in splenic B-cell responses with strong local germinal-center formation. Surprisingly, VLPs were not transported from the lung to the spleen in a free form but by B cells. The interaction between VLPs and B cells was initiated in the lung and occurred independently of complement receptor 2 and Fcγ receptors, but was dependent upon B-cell receptors. Thus, B cells passing through the lungs bind VLPs via their B-cell receptors and deliver them to local B cells within the splenic B-cell follicle. This process is fundamentally different from delivery of blood or lymph borne particulate antigens, which are transported into B cell follicles by binding to complement receptors on B cells.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Administração Intranasal , Transferência Adotiva , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/administração & dosagem , Antígenos Virais/sangue , Movimento Celular/imunologia , Feminino , Imunoglobulina G/biossíntese , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Complemento 3d/imunologia , Receptores de IgG/imunologia , Baço/imunologia , Baço/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/sangueRESUMO
A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies.
Assuntos
Anticorpos Antivirais/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Glicoproteínas de Membrana/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Switching de Imunoglobulina/efeitos dos fármacos , Switching de Imunoglobulina/genética , Imunoglobulina G/sangue , Imunoglobulina G/genética , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Pandemias , Transdução de Sinais/genética , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , VacinaçãoRESUMO
Dendritic cells (DCs) are the most important APC. It was recently reported that there is a dichotomy for Ag presentation by DC subsets; exogenous Ags reach the MHC class I pathway, but not the MHC class II pathway, in CD8(+) DCs, whereas CD8(-) DCs only process Ags for the MHC class II pathway. In this study, we used virus-like particles (VLPs) to show that CD8(+) and CD8(-) DCs efficiently capture and process VLPs for presentation in association with MHC class II in vivo. In contrast, CD8(+) DCs, but not CD8(-) DCs, cross presented VLP-derived peptides. This pattern was changed in an FcgammaR-dependent fashion in the presence of VLP-specific Abs, because under those conditions both DC subsets failed to efficiently cross present. Thus, the presentation of viral particles to CD4(+) T cells is not restricted to distinct DC subsets, whereas the presentation of viral particles to CD8(+) T cells is limited to CD8(+) DCs.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vírion/imunologia , Animais , Antígenos Virais/imunologia , Antígenos CD8/imunologia , Apresentação Cruzada/imunologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Innate stimuli, such as TLR ligands, are known to greatly facilitate cross-priming. Currently it is unclear whether innate stimuli enhance cross-priming at the level of cross-presentation or at the level of T-cell priming. In this study, we addressed this question by measuring cross-presentation as well as cross-priming by virus-like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus-like particles or using mice deficient in two key molecules of TLR-signaling, namely the adaptor molecule MyD88 as well as IFN-alpha/beta receptor. While efficient cross-presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand-mediated signals, T-cell priming was abolished. Thus, innate stimuli regulate cross-priming at the level of DC licensing for T-cell activation and not antigen presentation.
Assuntos
Apresentação Cruzada , Células Dendríticas/imunologia , Imunidade Inata , Transdução de Sinais , Animais , Apresentação de Antígeno , Células Dendríticas/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Proteínas Virais/imunologia , Vírion/imunologia , Vírion/ultraestruturaRESUMO
Viruses and virus-like particles (VLPs) are known to be potent inducers of B cell as well as Th cell and CTL responses. It is well established that professional APCs such as dendritic cells (DCs) and macrophages efficiently process viral particles for both MHC class I- and MHC class II-associated presentation, which is essential for induction of CTL and Th cell responses, respectively. Less is known, however, about the ability of B cells to present epitopes derived from viral particles to T cells. Using two different VLPs, in this study we show in vitro as well as in vivo that DCs present VLP-derived peptides in association with MHC class I as well as class II. In contrast, although B cells were able to capture VLPs similarly as DCs and although they efficiently processed VLPs for presentation in association with MHC class II, they failed to process exogenous VLPs for presentation in association with MHC class I. Thus, in contrast to DCs, B cells are not involved in the process of cross-priming. This finding is of physiological importance because B cells with the ability to cross-present Ag to specific CD8(+) T cells may be killed by these cells, preventing the generation of neutralizing Ab responses.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Apresentação Cruzada/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vírion/imunologia , Animais , Citotoxicidade Imunológica/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito B/imunologia , Citometria de Fluxo , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologiaRESUMO
The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.
Assuntos
Células Dendríticas/imunologia , Imunoglobulina A/biossíntese , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Mucosa/imunologia , RNA/imunologia , Animais , Glicoproteínas de Membrana , Camundongos , Camundongos Knockout , Linfócitos T Auxiliares-Indutores , Receptor 7 Toll-Like , Fator de Crescimento Transformador beta , Proteína Transmembrana Ativadora e Interagente do CAMLRESUMO
Due to their low immunogenicity in patients, humanized or fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infections, and immune disorders. Here, we describe a technology allowing for the rapid isolation of fully human mAbs. In contrast to previously described methods, B cells specific for an antigen of interest are directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant, antigen-specific single-chain Fv (scFv) libraries are generated from this pool of B cells and screened by mammalian cell surface display by using a Sindbis virus expression system. This method allows isolating antigen-specific antibodies by a single round of FACS. The variable regions (VRs) of the heavy chains (HCs) and light chains (LCs) are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this manner, several hypermutated high-affinity antibodies binding the Qbeta virus like particle (VLP), a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high expression levels in cell culture. The human nicotine-specific mAbs were validated preclinically in a mouse model. Thus, the technology presented here allows for rapid isolation of high-affinity, fully human antibodies with therapeutic potential from human volunteers.
Assuntos
Allolevivirus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Mamíferos , Biblioteca de Peptídeos , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Encéfalo/metabolismo , Proteínas do Capsídeo/imunologia , Citometria de Fluxo , Humanos , Imunização Passiva , Leucócitos Mononucleares/imunologia , Camundongos , Nicotina/imunologia , Sindbis virus/genética , Sindbis virus/metabolismoRESUMO
BACKGROUND: Allergen-specific desensitization (SIT) is the most effective therapy for allergies. Although allergen-specific antibodies have an important role in the process, mechanisms of IgG-mediated inhibition of allergic reactions are not well defined. OBJECTIVE: We investigated mechanisms by which SIT-induced allergen-specific IgGs inhibit allergic reactions. METHODS: We generated mAbs that recognize 3 nonoverlapping epitopes of the major cat allergen Fel d 1. Each of the mAbs was produced as an IgE and different IgG isotype. RESULTS: IgEs against 2 nonoverlapping epitopes on Fel d 1 are necessary and sufficient to sensitize mast cells for maximal FcepsilonRI signaling and degranulation on exposure to monomeric Fel d 1. IgE antibodies of a third specificity did not further increase mast cell degranulation, indicating that formation of large FcepsilonRI clusters are not required to induce maximal activation of mast cells. A single IgG that was specific for an epitope different from those recognized by the IgEs was a potent inhibitor of Fel d 1-mediated mast cell activation in vitro and in vivo. This inhibition required Fcgamma receptor-IIB. In human beings, IgGs of a single specificity were able to block degranulation of basophils from individuals with cat allergy. The inhibitory potential of these antibodies increased when larger allergen-IgG complexes were formed. CONCLUSIONS: These data reconcile conflicting theories in the literature and might explain the reason IgE levels do not necessarily decrease during therapy, despite clinical efficacy. These findings have important implications for vaccine design.
Assuntos
Alérgenos/imunologia , Anticorpos Monoclonais/farmacologia , Dessensibilização Imunológica , Glicoproteínas/imunologia , Hipersensibilidade/terapia , Imunoglobulina G/farmacologia , Alérgenos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/farmacologia , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de IgE/genética , Receptores de IgE/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Vacinas/genética , Vacinas/imunologiaRESUMO
T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.
Assuntos
Imunoglobulinas/fisiologia , Ativação Linfocitária/imunologia , Receptores de Complemento/fisiologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Antígeno B7-1/farmacologia , Antígeno B7-H1 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Imunoglobulinas/genética , Imunoglobulinas/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Lipopolissacarídeos/farmacologia , Fígado/citologia , Fígado/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miocardite/induzido quimicamente , Miocardite/imunologia , Miocardite/metabolismo , Peptídeos/farmacologia , Proteína 2 Ligante de Morte Celular Programada 1 , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tioglicolatos/farmacologiaRESUMO
Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.
Assuntos
Anticorpos Monoclonais , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Feminino , Humanos , Imunização Passiva , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Resultado do Tratamento , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Plasma cells are the main producers of antibody and key effector cells of the immune system. Despite their importance, analytics of plasma cells still suffers from the limited availability of specific markers. Currently, plasma cell identification relies on the expression of a single marker, CD138/syndecan-1. However, syndecan-1 is widely expressed on various cell types outside the hematopoietic compartment, and furthermore, not expressed on all subsets of plasma cells. To discover novel surface markers, a differential screening followed by signal sequence trap cloning was developed, leading to the identification of mouse Ly-6K (mLy-6K). Expression profiling confirmed that mLy-6K is expressed by plasma cells but not B cells or tissues not containing plasma cells. Expression at the surface of plasma cells isolated from spleen, lymph node, bone marrow, and lamina propria of the small intestine was demonstrated at the protein level using a polyclonal rabbit antibody. This novel plasma cell marker shows promise to help broaden our understanding of plasma cell differentiation and function.
Assuntos
Antígenos Ly/metabolismo , Plasmócitos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Ly/química , Antígenos Ly/genética , Biomarcadores/química , Biomarcadores/metabolismo , Northern Blotting , Linhagem Celular , Membrana Celular/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmócitos/citologia , Alinhamento de SequênciaRESUMO
Cell mediated immunity plays a vital role in defense against influenza infection in humans. Less is known about the role of vaccine-induced cell mediated immunity and the cytokine responses elicited. We measured CD4+ and CD8+ T-cell reactivity in human subjects following vaccination with licensed trivalent influenza vaccine and a novel virus-like particle based vaccine. We detected influenza-specific CD4+ T-cell responses following vaccination with the licensed trivalent influenza vaccine and found that these correlated with antibody measurements. Administration of the novel virus-like particle based vaccine elicited influenza-specific CD4+ and CD8+ T-cell responses and the induction of the cytokines IFN-γ, IL-17A, IL17F, IL-5, IL-13, IL-9, IL-10 and IL-21. Pre-existing cytokine responses influenced the profile of the cytokine response elicited by vaccination. In a subset of individuals the VLP vaccine changed pre-vaccination production of type 2 cytokines such as IL-5 and IL-13 to a post-vaccination type 1 cytokine signature characterized by IFN-γ. A transcriptional signature to vaccination was found to correlate with antibody titer, IFN-γ production by T-cells and expression of a putative RNA helicase, DDX17, on the surface of immune cells.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Citocinas/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Ativação Linfocitária/imunologia , Adulto , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Estudos de Coortes , Feminino , Humanos , Imunidade Celular/imunologia , Influenza Humana/imunologia , Influenza Humana/metabolismo , Masculino , Pessoa de Meia-Idade , Vacinação , Adulto JovemRESUMO
Since the discovery of the first virus-like particle (VLP) derived from hepatitis B virus in 1980 (1), the field has expanded substantially. Besides successful use of VLPs as safe autologous virus-targeting vaccines, the powerful immunogenicity of VLPs has been also harnessed to generate immune response against heterologous and even self-antigens (2-4). Linking adjuvants to VLPs displaying heterologous antigen ensures simultaneous delivery of all vaccine components to the same antigen-presenting cells. As a consequence, antigen-presenting cells, such as dendritic cells, will process and present the antigen displayed on VLPs while receiving costimulatory signals by the VLP-incorporated adjuvant. Similarly, antigen-specific B cells recognizing the antigen linked to the VLP are simultaneously exposed to the adjuvant. Here, we demonstrate in mice that physical association of antigen, carrier (VLPs), and adjuvant is more critical for B than T cell responses. As a model system, we used the E7 protein from human papilloma virus, which spontaneously forms oligomers with molecular weight ranging from 158 kDa to 10 MDa at an average size of 50 nm. E7 oligomers were either chemically linked or simply mixed with VLPs loaded with DNA rich in non-methylated CG motifs (CpGs), a ligand for toll-like receptor 9. E7-specific IgG responses were strongly enhanced if the antigen was linked to the VLPs. In contrast, both CD4+ and CD8+ T cell responses as well as T cell-mediated protection against tumor growth were comparable for linked and mixed antigen formulations. Therefore, our data show that B cell but not T cell responses require antigen-linkage to the carrier and adjuvant for optimal vaccination outcome.
RESUMO
Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.
Assuntos
Cistatinas/metabolismo , Doenças Priônicas/metabolismo , Animais , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patologia , Cistatinas/líquido cefalorraquidiano , Cistatinas/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Doenças Priônicas/líquido cefalorraquidiano , Doenças Priônicas/genética , Doenças Priônicas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
T cell responses are regulated by the affinity/avidity of the T cell receptor for the MHC/peptide complex, available costimulation and duration of antigenic stimulation. Altered peptide ligands (APLs) are usually recognized with a reduced affinity/avidity by the T cell receptor and are often able to only partially activate T cells in vitro or may even function as antagonists. Here we assessed the ability of APLs derived from peptide p33 of lymphocytic choriomeningitis virus (LCMV) to mediate lysis of target cells in vivo, confer anti-viral protection and cause auto-immune disease. In general, in vitro cross-reactivity between APLs was rather limited, and even strongly cross-reactive cytotoxic T lymphocytes were only able to mediate moderate anti-viral protection. Partial protection was observed for infection with LCMV or low doses of recombinant vaccinia virus, while no reduced viral titers could be seen upon infection with high dose of vaccinia virus. In a transgenic mouse model expressing LCMV glycoprotein in the islets of the pancreas, APLs induced a transient insulitis but failed to induce autoimmune diabetes. Thus, effector functions induced by even highly homologous APLs are rather limited in vivo.
Assuntos
Antígenos Virais/farmacologia , Glicoproteínas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/farmacologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/prevenção & controle , Reações Cruzadas , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Modelos Animais de Doenças , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/imunologia , Ligantes , Coriomeningite Linfocítica/complicações , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Vaccinia virus/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Alphavirus vectors are attractive as recombinant protein expression systems due to the high level of gene expression achieved. The combination of two mutations in the viral replicase, which render the replicase noncytopathic and temperature-sensitive, allowed the generation of a DNA-based vector (CytTs) that shows temperature inducible expression. This vector is of significant value for the production of toxic protein. However, like for other stable expression systems, tedious screening of individual cell clones are required in order to get a high producer cell clone. To circumvent this, we generated an episomally replicating vector by introducing an Epstein-Barr virus mini-replicon unit into CytTs. This novel vector allowed rapid generation of cell populations that showed tight regulation of expression and comparable expression levels to the ones achieved with high producer cell clones with CytTs. Moreover, protein production with selected cell populations could easily be scaled-up to a fermentation process.