RESUMO
More than 3 years into the global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat to public health. Immunities acquired from infection or current vaccines fail to provide long term protection against subsequent infections, mainly due to their fast-waning nature and the emergence of variants of concerns (VOCs) such as Omicron. To overcome these limitations, SARS-CoV-2 Spike protein receptor binding domain (RBD)-based epitopes are investigated as conjugates with a powerful carrier, the mutant bacteriophage Qß (mQß). The epitope design is critical to eliciting potent antibody responses with the full length RBD being superior to peptide and glycopeptide antigens. The full length RBD conjugated with mQß activates both humoral and cellular immune systems in vivo, inducing broad spectrum, persistent, and comprehensive immune responses effective against multiple VOCs including Delta and Omicron variants, rendering it a promising vaccine candidate.
Assuntos
Linfócitos B , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Linfócitos T , SARS-CoV-2/imunologia , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Linfócitos T/imunologia , Animais , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Linfócitos B/imunologia , Camundongos , Humanos , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/química , Mutação , Feminino , Allolevivirus/imunologia , Allolevivirus/química , Anticorpos Antivirais/imunologia , Domínios Proteicos , Camundongos Endogâmicos BALB C , Imunidade CelularRESUMO
The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qß conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qß), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qß-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qß conjugates synthesized represent a promising candidate of HIV-1 vaccine.
Assuntos
Allolevivirus/imunologia , Glicopeptídeos/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/uso terapêutico , Anticorpos Neutralizantes/imunologia , Antígenos/imunologia , Epitopos/genética , Epitopos/imunologia , Glicopeptídeos/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fagocitose/imunologiaRESUMO
The first synthetic carbohydrate based potential anti-Salmonella Enteritidis vaccine has been developed by conjugating a synthetic tetrasaccharide antigen with bacteriophage Qß. High levels of specific and long lasting anti-glycan IgG antibodies were induced by the conjugate, which completely protected mice from lethal bacterial challenges in a passive transfer model.
Assuntos
Antígenos O/metabolismo , Oligossacarídeos/síntese química , Vacinas contra Salmonella/imunologia , Salmonella enteritidis/metabolismo , Allolevivirus/química , Allolevivirus/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/uso terapêutico , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Camundongos , Antígenos O/química , Antígenos O/imunologia , Oligossacarídeos/química , Oligossacarídeos/imunologia , Salmonelose Animal/mortalidade , Salmonelose Animal/prevenção & controle , Salmonelose Animal/terapia , Salmonella enteritidis/patogenicidade , Taxa de Sobrevida , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/imunologiaRESUMO
Although TLR signaling in B cells has been implicated in the germinal center (GC) responses during viral infections and autoimmune diseases, the underlying mechanism is unclear. Bacterial phage Qß-derived virus-like particle (Qß-VLP) contains TLR ligands, which can enhance Qß-VLP-induced Ab response, including GC response, through TLR/MyD88 signaling in B cells. In this study, by examining Ag-specific B cell response to Qß-VLP, we found that lack of B cell MyD88 from the beginning of the immune response led to a more severe defect in the GC scale than abolishing MyD88 at later time points of the immune response. Consistently, B cell-intrinsic MyD88 signaling significantly enhanced the initial proliferation of Ag-specific B cells, which was accompanied with a dramatic increase of plasma cell generation and induction of Bcl-6+ GC B cell precursors. In addition, B cell-intrinsic MyD88 signaling promoted strong T-bet expression independent of IFN-γ and led to the preferential isotype switching to IgG2a/c. Thus, by promoting the initial Ag-specific B cell proliferation and differentiation, B cell-intrinsic MyD88 signaling enhanced both T-independent and T-dependent Ab responses elicited by Qß-VLP. This finding will provide additional insight into the role of TLR signaling in antiviral immunity, autoimmune diseases, and vaccine design.
Assuntos
Allolevivirus/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptores Toll-Like/imunologia , Animais , Anticorpos Antivirais/imunologia , Diferenciação Celular/imunologia , Proliferação de Células/fisiologia , Feminino , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais/imunologia , Proteínas com Domínio T/biossíntese , Proteínas Estruturais Virais/imunologiaRESUMO
Tumor-associated carbohydrate antigens (TACAs) are attractive targets for anticancer vaccine development. Due to the low immunogenicity of TACAs, a powerful carrier system is needed to boost immune responses. Virus-like particles (VLPs) are an exciting platform for delivering TACAs to the immune system. The high symmetry of VLPs enables the display of TACAs in an organized manner, which in turn can potently activate antibody secreting B cells, eliciting high titers of antiglycan IgG antibodies. In this chapter, the protocol for conjugating a prototypical TACA, the Tn antigen to a VLP, bacteriophage Qß, is presented. On an average around 370 copies of Tn can be attached to each Qß capsid. Immunization of mice with Qß-Tn conjugate leads to over two orders of magnitude higher IgG antibodies compared to control mice receiving Qß only without the Tn antigen. Antibodies induced by Qß-Tn recognize Tn-expressing tumor cells strongly and protect mice from tumor-induced death. The techniques for evaluating antibody titers by enzyme-linked immunosorbent assay, antibody binding to tumor cells by flow cytometry, and the protection efficacy of the vaccine in a therapeutic model of tumor are discussed in this chapter.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias/terapia , Vacinas Conjugadas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Allolevivirus/imunologia , Animais , Antígenos Glicosídicos Associados a Tumores/uso terapêutico , Linfócitos B/imunologia , Biomarcadores Tumorais/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Imunização/métodos , Camundongos , Neoplasias/imunologia , Vacinas Conjugadas/uso terapêutico , Vacinas de Partículas Semelhantes a Vírus/uso terapêuticoRESUMO
Viral nanoparticles have been utilized as a platform for vaccine development and are a versatile system for the display of antigenic epitopes for a variety of disease states. However, the induction of a clinically relevant immune response often requires multiple injections over an extended period of time, limiting patient compliance. Polymeric systems to deliver proteinaceous materials have been extensively researched to provide sustained release, which would limit administration to a single dose. Melt-processing is an emerging manufacturing method that has been utilized to create polymeric materials laden with proteins as an alternative to typical solvent-based production methods. Melt-processing is advantageous because it is continuous, solvent-free, and 100% of the therapeutic protein is encapsulated. In this study, we utilized melt-encapsulation to fabricate viral nanoparticle laden polymeric materials that effectively deliver intact particles and generate carrier specific antibodies in vivo. The effects of initial processing and postprocessing on particle integrity and aggregation were studied to develop processing windows for scale-up and the creation of more complex materials. The dispersion of particles within the PLGA matrix was studied, and the effect of additives and loading level on the release profile was determined. Overall, melt-encapsulation was found to be an effective method to produce composite materials that can deliver viral nanoparticles over an extended period and elicit an immune response comparable to typical administration schedules.
Assuntos
Implantes Absorvíveis , Allolevivirus/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Vírion/química , Allolevivirus/imunologia , Animais , Imunização/métodos , Masculino , Camundongos Endogâmicos BALB C , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vírion/imunologiaRESUMO
DNA rich in unmethylated CG motifs (CpGs) engage Toll-Like Receptor 9 (TLR-9) in endosomes and are well described stimulators of the innate and adaptive immune system. CpGs therefore can efficiently improve vaccines' immunogenicity. Packaging CpGs into nanoparticles, in particular into virus-like particles (VLPs), improves the pharmacological characteristics of CpGs as the protein shell protects them from DNAse activity and delivers the oligomers to the endosomal compartments of professional antigen presenting cells (APCs). The current consensus in packaging and delivering CpGs in VLP-based vaccines is that both adjuvants and antigens should be kept in close proximity (i.e. physically linked) to ensure delivery of antigens and adjuvants to the same APCs. In the current study, we harness the draining properties of the lymphatic system and show that also non-linked VLPs are efficiently co-delivered to the same APCs in lymph nodes. Specifically, we have shown that CpGs can be packaged in one VLP and mixed with another VLP displaying the antigen prior to administration in vivo. Both VLPs efficiently reached the same draining lymph node where they were taken up and processed by the same APCs, namely dendritic cells and macrophages. This resulted in induction of specific CTLs producing cytokines and killing target cells in vivo at levels seen when using VLPs containing both CpGs and chemically conjugated antigen. Thus, delivery of antigens and adjuvants in separate nanoparticles eliminates the need of physical conjugation and thus can be beneficial when designing precision medicine VLP-based vaccines or help to re-formulate existing VLP vaccines not naturally carrying immunostimulatory sequences.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos Virais/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Allolevivirus/imunologia , Animais , Antígenos Virais/imunologia , Ilhas de CpG , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Sistemas de Liberação de Medicamentos , Linfonodos/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/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
Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. For a substantial proportion of patients, conventional drug treatments do not provide adequate pain relief. Consequently, novel approaches to pain management, involving alternative targets and new therapeutic modalities compatible with chronic use, are being sought. Nerve growth factor (NGF) is a major mediator of chronic pain. Clinical testing of NGF antagonists is ongoing, and clinical proof of concept has been established with a neutralizing mAb. Active immunization, with the goal of inducing therapeutically effective neutralizing autoreactive Abs, is recognized as a potential treatment option for chronic diseases. We have sought to determine if such a strategy could be applied to chronic pain by targeting NGF with a virus-like particle (VLP)-based vaccine. A vaccine comprising recombinant murine NGF conjugated to VLPs from the bacteriophage Qß (NGFQß) was produced. Immunization of mice with NGFQß induced anti-NGF-specific IgG Abs capable of neutralizing NGF. Titers could be sustained over 1 y by periodic immunization but declined in the absence of boosting. Vaccination with NGFQß substantially reduced hyperalgesia in collagen-induced arthritis or postinjection of zymosan A, two models of inflammatory pain. Long-term NGFQß immunization did not change sensory or sympathetic innervation patterns or induce cholinergic deficits in the forebrain, nor did it interfere with blood-brain barrier integrity. Thus, autovaccination targeting NGF using a VLP-based approach may represent a novel modality for the treatment of chronic pain.
Assuntos
Hiperalgesia/imunologia , Hiperalgesia/prevenção & controle , Mediadores da Inflamação/uso terapêutico , Fatores de Crescimento Neural/imunologia , Manejo da Dor , Dor/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Doença Aguda , Allolevivirus/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/fisiologia , Anticorpos Antivirais/uso terapêutico , Linhagem Celular Tumoral , Doença Crônica , Avaliação Pré-Clínica de Medicamentos , Hiperalgesia/virologia , Inflamação/imunologia , Inflamação/patologia , Inflamação/prevenção & controle , Mediadores da Inflamação/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fatores de Crescimento Neural/efeitos adversos , Fatores de Crescimento Neural/uso terapêutico , Testes de Neutralização , Dor/patologia , Ratos , Fatores de Tempo , Vacinas Conjugadas/efeitos adversos , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêutico , Vacinas de Partículas Semelhantes a Vírus/efeitos adversos , Vacinas de Partículas Semelhantes a Vírus/uso terapêuticoRESUMO
The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the "glycan shield" defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Q beta and cowpea mosaic virus (CPMV). The Q beta glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV.
Assuntos
Vacinas contra a AIDS/química , Allolevivirus/imunologia , Anticorpos Monoclonais/imunologia , Capsídeo/imunologia , Comovirus/imunologia , HIV/imunologia , Manose/imunologia , Vacinas contra a AIDS/imunologia , Allolevivirus/química , Animais , Anticorpos Amplamente Neutralizantes , Capsídeo/química , Sequência de Carboidratos , Comovirus/química , Anticorpos Anti-HIV , Infecções por HIV/prevenção & controle , Manose/química , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/imunologia , CoelhosRESUMO
IL-21 produced by follicular Th (Tfh) cells is an important regulator of Tfh cell development and B cell responses, including germinal center (GC) formation. However, whether defective GC formation and Ab responses are a consequence of impaired Tfh cells development or a B cell-intrinsic defect in IL-21-deficient mice requires clarification. To address this question, we generated chimeric mice lacking IL-21R exclusively on B cells. In this study, we demonstrate that GC reaction and B cell responses induced by immunization with virus-like particles were strongly reduced in both global and B cell-specific IL-21R-deficient mice. Interestingly, the presence of TLR7 ligand within virus-like particles largely restored defective GC reaction and Ab responses in global as well as in B cell-specific IL-21R-deficient mice. Hence, IL-21 acts directly on B cells and cooperates with TLR signaling for optimal B cell responses.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interleucinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/fisiologia , Receptor 8 Toll-Like/fisiologia , Allolevivirus/genética , Allolevivirus/imunologia , Animais , Anticorpos Antivirais/biossíntese , Subpopulações de Linfócitos B/virologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Regulação Viral da Expressão Gênica/imunologia , Produtos do Gene env/biossíntese , Produtos do Gene env/genética , Produtos do Gene env/fisiologia , Centro Germinativo/citologia , Centro Germinativo/virologia , Imunidade Inata/genética , Imunoglobulina G/biossíntese , Interleucinas/metabolismo , Ligantes , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-21/deficiência , Receptores de Interleucina-21/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/virologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Vírion/genética , Vírion/imunologiaRESUMO
Interleukin-5 (IL-5) is a cytokine which is essential for the maturation of eosinophils in bone marrow and for their release into the blood. Eotaxin is a CC type chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders. Since eosinophil-activity is governed by these two pathways, we targeted both IL-5 and eotaxin by active vaccination to block eosinophilia. We produced two vaccines by chemically cross-linking IL-5 or eotaxin to a virus-like particle (VLP) derived from the bacteriophage Qbeta, yielding highly repetitive arrays of these cytokines on the VLP surface. Both vaccines overcame self-tolerance and induced high antibody titers against the corresponding self-molecules in mice. Immunization with either of the two vaccines reduced eosinophilic inflammation of the lung in an ovalbumin (OVA) based mouse model of allergic airway inflammation. Animals immunized with the two vaccines at the same time developed high antibody titers against both cytokines and also reduced eosinophil-infiltration of the lung. These data demonstrate that targeting either IL-5 or eotaxin may lower eosinophilia. Simultaneous immunization against IL-5 and eotaxin demonstrates that such a therapeutic approach may be used to treat complex disorders in which multiple mediators are involved.
Assuntos
Allolevivirus/imunologia , Quimiocina CCL11/imunologia , Eosinofilia/prevenção & controle , Interleucina-5/imunologia , Vacinação/métodos , Proteínas Virais/imunologia , Alérgenos/imunologia , Animais , Autoanticorpos/sangue , Feminino , Hipersensibilidade/patologia , Hipersensibilidade/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Doenças Respiratórias/imunologia , Doenças Respiratórias/patologia , Vacinas Virossomais/imunologiaRESUMO
Pre-existing immunity against vaccine carrier proteins has been reported to inhibit the immune response against antigens conjugated to the same carrier by a process termed carrier induced epitopic suppression (CIES). Hence understanding the phenomenon of CIES is of major importance for the development of conjugate vaccines. Virus-like particles (VLPs) are a novel class of potent immunological carriers which have been successfully used to enhance the antibody response to virtually any conjugated antigen. In the present study we investigated the impact of a pre-existing VLP-specific immune response on the development of antibody responses against a conjugated model peptide after primary, secondary and tertiary immunization. Although VLP-specific immune responses led to reduced peptide-specific antibody titers, we showed that CIES against peptide-VLP conjugates could be overcome by high coupling densities, repeated injections and/or higher doses of conjugate vaccine. Furthermore we dissected VLP-specific immunity by adoptively transferring VLP-specific antibodies, B-cells or T(helper) cells separately into naïve mice and found that the observed CIES against peptide-VLP conjugates was mainly mediated by carrier-specific antibodies.
Assuntos
Allolevivirus/imunologia , Anticorpos Antivirais/imunologia , Antígenos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Epitopos/imunologia , Tolerância Imunológica , Peptídeos/imunologia , Allolevivirus/genética , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Linfócitos T Auxiliares-Indutores/imunologia , VirossomosRESUMO
Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qbeta-derived virus-like particles (Qbeta-Fel d1). Qbeta-Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcgammaRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcgammaRIIb. In addition, treatment with Qbeta-Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qbeta-Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qbeta-Fel d1 may be a safe and effective treatment for cat allergy.
Assuntos
Allolevivirus/imunologia , Glicoproteínas/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/imunologia , Vacinas/imunologia , Análise de Variância , Animais , Anticorpos Monoclonais , Teste de Degranulação de Basófilos , Gatos , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genéticaRESUMO
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
IL-1 is an important mediator of inflammation and a major cause of tissue damage in rheumatoid arthritis (RA). Therapeutic administration of recombinant IL-1 receptor antagonist (IL-1Ra) is efficacious in reducing clinical symptoms of disease, but suffers from several drawbacks, including the need for frequent administrations of large amounts. Here, we show that immunization of mice with either IL-1alpha or IL-1beta chemically cross-linked to virus-like particles (VLP) of the bacteriophage Qbeta elicited a rapid and long-lasting autoantibody response. The induced Ab efficiently neutralized the binding of the respective IL-1 molecules to their receptors in vitro and their pro-inflammatory activities in vivo. In the collagen-induced arthritis model, both vaccines strongly protected mice from inflammation and degradation of bone and cartilage. Moreover, immunization with either vaccine showed superior efficacy than daily administrations of high amounts of IL-1Ra. In the T and B cell-independent collagen Ab transfer model, immunization with the IL-1beta vaccine strongly protected from arthritis, whereas immunization with the IL-1alpha vaccine had no effect. Our results suggest that active immunization with IL-1alpha, and especially IL-1beta conjugated to Qbeta VLP, might become an efficacious and cost-effective new treatment option for RA and other systemic IL-1-dependent inflammatory disorders.
Assuntos
Allolevivirus/imunologia , Artrite Experimental/prevenção & controle , Doenças Autoimunes/prevenção & controle , Interleucina-1/uso terapêutico , Vacinas de Subunidades Antigênicas/uso terapêutico , Aminoácidos/sangue , Animais , Formação de Anticorpos/imunologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Colágeno Tipo II/imunologia , Reações Cruzadas/imunologia , Feminino , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-1alfa/genética , Interleucina-1alfa/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Testes de Neutralização , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Vacinação/métodos , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Neutralization of the proinflammatory cytokine TNF-alpha by mAbs or soluble receptors represents an effective treatment for chronic inflammatory disorders such as rheumatoid arthritis, psoriasis, or Crohn's disease. In this study, we describe a novel active immunization approach against TNF-alpha, which results in the induction of high titers of therapeutically active autoantibodies. Immunization of mice with virus-like particles of the bacteriophage Qbeta covalently linked to either the entire soluble TNF-alpha protein (Qbeta-C-TNF(1-156)) or a 20-aa peptide derived from its N terminus (Qbeta-C-TNF(4-23)) yielded specific Abs, which protected from clinical signs of inflammation in a murine model of rheumatoid arthritis. Whereas mice immunized with Qbeta-C-TNF(1-156) showed increased susceptibility to Listeria monocytogenes infection and enhanced reactivation of latent Mycobacterium tuberculosis, mice immunized with Qbeta-C-TNF(4-23) were not immunocompromised with respect to infection with these pathogens. This difference was attributed to recognition of both transmembrane and soluble TNF-alpha by Abs elicited by Qbeta-C-TNF(1-156), and a selective recognition of only soluble TNF-alpha by Abs raised by Qbeta-C-TNF(4-23). Thus, by specifically targeting soluble TNF-alpha, Qbeta-C-TNF(4-23) immunization has the potential to become an effective and safe therapy against inflammatory disorders, which might overcome the risk of opportunistic infections associated with the currently available TNF-alpha antagonists.
Assuntos
Allolevivirus/imunologia , Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/prevenção & controle , Feminino , Listeriose/imunologia , Listeriose/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Dados de Sequência Molecular , Tamanho da Partícula , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/imunologia , Engenharia de Proteínas , Índice de Gravidade de Doença , Solubilidade , Tuberculose/diagnóstico , Tuberculose/prevenção & controle , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/deficiência , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/efeitos adversos , Vacinas Conjugadas/imunologia , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/efeitos adversos , Vacinas Virossomais/imunologiaRESUMO
Protective Ab levels can be maintained for years upon infection or vaccination. In this study, we studied the duration of Ab responses as a function of the life span of plasma cells and tested the role of persisting Ag in maintaining B cell memory. Our analysis of B cell responses induced in mice immunized with virus-like particles demonstrates the following: 1) Ab titers are long-lived, but decline continuously with a t(1/2) of approximately 80 days, which corresponds to the life span of plasma cells; 2) the germinal center (GC) reaction, which lasts for up to 100 days, is dependent on Ag associated with follicular dendritic cells; and 3) early GCs produce massive numbers of plasma and memory B cell precursors, whereas the late Ag-dependent GCs are dispensable for the maintenance of Ab levels and B cell memory.
Assuntos
Anticorpos/sangue , Antígenos/imunologia , Memória Imunológica , Plasmócitos/imunologia , Allolevivirus/imunologia , Animais , Antígenos/sangue , Proliferação de Células , Células Dendríticas Foliculares/efeitos dos fármacos , Células Dendríticas Foliculares/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Imunização , Imunoglobulinas/farmacologia , Terapia de Imunossupressão , Cinética , Receptor beta de Linfotoxina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/citologia , Vírion/imunologiaRESUMO
A vaccine targeting the amyloid-beta (Abeta) peptide is a promising potential immunotherapy for Alzheimer's disease patients. However, experience from a recent clinical trial of a candidate Abeta vaccine has suggested that it is important to develop techniques to induce high titer antibodies against Abeta associated with vaccine efficacy while reducing the T cell responses against Abeta that were potentially responsible for serious side effects. We have previously demonstrated that immunization with self- and foreign antigens arrayed in a repetitive fashion on the surface of virus-like particles (VLPs) induces high titer antibody responses at low doses and in the absence of potentially inflammatory adjuvants. In this study, we examined the antibody and T cell responses upon immunization with human papillomavirus VLP- and Qbeta bacteriophage-based Abeta vaccines. Immunization with Abeta conjugated to VLPs or Qbeta elicited anti-Abeta antibody responses at low doses and without the use of adjuvants. The flexibility of these virus-based display systems allowed us to link and induce antibodies against short Abeta-derived peptides from the amino- and carboxyl-termini of the peptide. Immunization of mice with Abeta peptide in combination with Freund's adjuvant elicited predominantly IgG2c antibodies and strong T cell proliferative responses against Abeta. In contrast, VLP-conjugated Abeta peptides elicited more balanced isotype responses, dominated by IgG1. Both VLP and Qbeta-based Abeta vaccines induced weak or negligible T cell responses against Abeta. T cell responses were largely directed against linked viral epitopes. Taken together, virus-based vaccines that allow the presentation of Abeta in a repetitive dense array are new and potentially more effective vaccine candidates for Alzheimer's disease.
Assuntos
Allolevivirus/imunologia , Doença de Alzheimer/imunologia , Vacinas contra Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Papillomavirus Humano 16/imunologia , Linfócitos T/imunologia , Doença de Alzheimer/virologia , Animais , Formação de Anticorpos/imunologia , Especificidade de Anticorpos , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: In mice, highly repetitive antigens, such as those present on bacterial or viral surfaces, efficiently cross-link B-cell receptors and therefore induce strong IgG responses. In this study we covalently coupled a synthetic 16-amino-acid sequence of the allergen Der p 1 to a virus-like particle derived from the bacteriophage Qbeta (Qbeta-Der p 1). OBJECTIVE: We evaluated the safety and immunogenicity of Qbeta-Der p 1 in human subjects and compared different doses and routes of immunization. METHODS: In a phase I trial 24 healthy volunteers were randomly assigned to one of 4 treatment groups. Group 1 received 50 microg of Qbeta-Der p 1 intramuscularly, group 2 received 50 microg of Qbeta-Der p 1 subcutaneously, group 3 received 10 microg of Qbeta-Der p 1 intramuscularly, and group 4 received 10 microg of Qbeta-Der p 1 subcutaneously. Boosting immunizations with 10 microg were given after 1 and 3 months. Antibody titers were measured after 1, 3, 4, 6, 12, and 18 months. RESULTS: The vaccine Qbeta-Der p 1 was well tolerated. Significant IgG responses were observed 4 weeks after a single injection. Individuals receiving 50 microg of the vaccine had significantly higher IgG titers than those vaccinated with 10 microg. However, the route of immunization (subcutaneous vs intramuscular) had no effect. In the 50-microg dose group, strong antibody responses against Der p 1 with average titers of 1:2000 were obtained. CONCLUSION: Vaccination with a peptide antigen covalently coupled to highly repetitive virus-like particles represents an adjuvant-free means of rapidly inducing high antibody titers in human subjects. CLINICAL IMPLICATIONS: Allergens coupled to virus-like particles can be used to enhance the efficiency of allergen-specific immunotherapy.