RESUMO
IL-2 is critical for peripheral tolerance mediated by regulatory T (Treg) cells, which represent an obstacle for effective cancer immunotherapy. Although IL-2 is important for effector (E) T cell function, it has been hypothesized that therapies blocking IL-2 signals weaken Treg cell activity, promoting immune responses. This hypothesis has been partially tested using anti-IL-2 or anti-IL-2R Abs with antitumor effects that cannot be exclusively attributed to lack of IL-2 signaling in vivo. In this work, we pursued an alternative strategy to block IL-2 signaling in vivo, taking advantage of the trimeric structure of the IL-2R. We designed an IL-2 mutant that conserves the capacity to bind to the αß-chains of the IL-2R but not to the γc-chain, thus having a reduced signaling capacity. We show our IL-2 mutein inhibits IL-2 Treg cell-dependent differentiation and expansion. Moreover, treatment with IL-2 mutein reduces Treg cell numbers and impairs tumor growth in mice. A mathematical model was used to better understand the effect of the mutein on Treg and E T cells, suggesting suitable strategies to improve its design. Our results show that it is enough to transiently inhibit IL-2 signaling to bias E and Treg cell balance in vivo toward immunity.
Assuntos
Proliferação de Células/efeitos dos fármacos , Interleucina-2/antagonistas & inibidores , Linfocinas/farmacologia , Neoplasias/terapia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/metabolismo , Tolerância Periférica/efeitos dos fármacosRESUMO
A mutein with stronger antitumor activity and lower toxicity than wild-type human interleukin-2 (IL-2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL-2 receptor alpha chain (thought to be critical for both IL-2-driven expansion of T regulatory cells and IL-2-mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL-2-derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage-displayed IL-2 variants to induce proliferation of the IL-2-dependent CTLL-2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display-based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL-2 and related cytokines, accelerating the development of cytokine-derived therapeutics.
Assuntos
Bacteriófagos/metabolismo , Interleucina-2/química , Linfotoxina-alfa/química , Proteínas Recombinantes/química , Proliferação de Células , HumanosRESUMO
IL-2 has been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy and severe toxicity. Currently, it is assumed that part of the limited efficacy is due to the IL-2-driven preferential expansion of regulatory T cells, which dampen the antitumor immunity. In this study, we characterize a human IL-2 mutant with higher antitumor efficacy and lower toxicity than wild type human IL-2 (wtIL-2). The mutant differs from wtIL-2 by four mutations at the interface with the α subunit of IL-2R. The IL-2 mutant induces in vitro proliferation of CD8(+)CD44(hi) and NK1.1 cells as efficiently as does wtIL-2, but it shows a reduced capacity to induce proliferation of CD4(+)Foxp3(+) regulatory T cells. The IL-2 mutant shows a higher antimetastatic effect than does wtIL-2 in several transplantable tumor models: the experimental metastasis model of MB16F0 melanoma and the experimental and spontaneous metastasis models for the mouse pulmonary carcinoma 3LL-D1222. Relevantly, the IL-2 mutant also exhibits lower lung and liver toxicity than does wtIL-2 when used at high doses in mice. In silico simulations, using a calibrated mathematical model, predict that the properties of IL-2 mutein are a consequence of the reduction, of at least two orders of magnitude, in its affinity for the α subunit of IL-2R (CD25). The human IL-2 mutant described in the present work could be a good candidate for improving cancer therapy based on IL-2.
Assuntos
Imunoterapia/métodos , Interleucina-2/genética , Interleucina-2/imunologia , Neoplasias Experimentais/terapia , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Interleucina-2/química , Melanoma/imunologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Teóricos , Mutação , Neoplasias Experimentais/imunologia , Estrutura Quaternária de ProteínaRESUMO
Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.
RESUMO
The current chapter focuses on the use of filamentous phages to display and modify biologically active cytokines, with special emphasis on directed evolution of novel variants showing improved receptor binding. Cytokines are essential protein mediators involved in cell-to-cell communication. Their functional importance and the complexity of their interactions with multichain receptors make cytokine engineering a promising tool for the discovery and optimization of therapeutic molecules. Protocols used at the laboratory are illustrated through examples of manipulation of interleukin-2 and interleukin-6, two members of the family of alpha-helix-bundle cytokines playing pivotal roles in immunity and inflammation.
Assuntos
Bacteriófagos , Citocinas , Humanos , Interleucina-6 , Comunicação Celular , InflamaçãoRESUMO
BACKGROUND: In a previous work, an IL-2Rßγ biased mutant derived from human IL-2 and called IL-2noα, was designed and developed. Greater antitumor effects and lower toxicity were observed compared to native IL-2. Nevertheless, mutein has some disadvantages, such as a very short half-life of about 9-12 min, propensity for aggregation, and solubility problems. OBJECTIVE: In this study, PEGylation was employed to improve the pharmacokinetic and antitumoral properties of the novel protein. METHODS: Pegylated IL-2noα was characterized by polyacrylamide gel electrophoresis, size exclusion chromatography, in vitro cell proliferation and in vivo cell expansion bioassays, and pharmacokinetic and antitumor studies. RESULTS: IL-2noα-conjugates with polyethylene glycol (PEG) of 1.2 kDa, 20 kDa, and 40 kDa were obtained by classical acylation. No significant changes in the secondary and tertiary structures of the modified protein were detected. A decrease in biological activity in vitro and a significant improvement in half-life were observed, especially for IL-2noα-PEG20K. PEGylation of IL-2noα with PEG20K did not affect the capacity of the mutant to induce preferential expansion of T effector cells over Treg cells. This pegylated IL-2noα exhibited a higher antimetastatic effect compared to unmodified IL-2noα in the B16F0 experimental metastases model, even when administered at lower doses and less frequently. CONCLUSION: PEG20K was selected as the best modification strategy, to improve the blood circulation time of the IL-2noα with a superior antimetastatic effect achieved with lower doses.
Assuntos
Interleucina-2 , Proteínas , Humanos , Polietilenoglicóis/químicaRESUMO
Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.
Assuntos
Evolução Molecular Direcionada , Subunidade beta de Receptor de Interleucina-2 , Interleucina-2 , Biblioteca de Peptídeos , Humanos , Subunidade beta de Receptor de Interleucina-2/química , Interleucina-2/química , Interleucina-2/genética , Interleucina-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Evolução Molecular Direcionada/métodos , Domínios Proteicos , Animais , Camundongos , Linhagem Celular TumoralRESUMO
PURPOSE: The F3II cell line is a highly invasive variant of mammary carcinoma. Although it is frequently used as a model to evaluate the efficacy of immunotherapy, its impact on the immune system remains poorly understood. The main objectives of this study were to evaluate the effects of F3II tumors on the development of chronic inflammation and to characterize tumor-associated immunosuppression. METHODS: Following the experimental implantation of F3II tumors in BALB/c mice, alterations in the liver and spleen anatomy and the numbers of circulating leukocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells were measured using hematological techniques, histopathological analysis, and flow cytometry. The capacity of the F3II tumor-bearing mice to reject MB16F10 allogeneic tumor transplantation was also evaluated. In addition, the restoration of immune parameters in tumor-bearing mice was evaluated after standard breast cancer chemotherapy and surgical tumor excision. RESULTS: F3II tumor implantation increased the levels of chronic inflammatory markers, such as the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios, and caused myeloid alterations, including extramedullary granulopoiesis and megakaryopoiesis, along with the recruitment of MDSCs to the spleen. Chemotherapy or surgical F3II tumor removal completely rescued the tumor-associated extramedullary granulopoiesis and megakaryopoiesis. Notably, the presence of F3II tumors reduced the capacity of BALB/c mice to reject MB16F10 allogeneic tumor transplantation. CONCLUSION: These results support the occurrence of F3II tumor-mediated immune cell dysfunction, which mimics the immune alterations characterized by chronic systemic inflammation and immunosuppression observed in breast cancer in clinical settings. Thus, the F3II tumor model is relevant for evaluating novel breast cancer immunotherapies and combinations in preclinical studies. This model could also be useful for identifying appropriate therapeutic targets and developing proof-of-concept experiments in the future.
RESUMO
High doses of interleukin-2 (IL-2) have been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy, with a ~15% response rate. Remarkably, 7%-9% of patients achieve complete or long-lasting responses. Many patients treated with IL-2 experienced an expansion of regulatory T cells (Tregs), specifically the expansion of ICOS+ highly suppressive Tregs, which correlate with worse clinical outcomes. This partial efficacy together with the high toxicity associated with the therapy has limited the use of IL-2-based therapy. Taking into account the understanding of IL-2 structure, signaling, and in vivo functions, some efforts to improve the cytokine properties are currently under study. In previous work, we described an IL-2 mutein with higher antitumor activity and less toxicity than wtIL-2. Mutein was in silico designed for losing the binding capacity to CD25 and for preferential stimulation of effector cells CD8+ and NK cells but not Tregs. Mutein induces a higher anti-metastatic effect than wtIL-2, but the extent of the in vivo antitumor activity was still unexplored. In this work, it is shown that mutein induces a strong antitumor effect on four primary tumor models, being effective even in those models where wtIL-2 does not work. Furthermore, mutein can change the in vivo balance between Tregs and T CD8+ memory/activated cells toward immune activation, in both healthy and tumor-bearing mice. This change reaches the tumor microenvironment and seems to be the major explanation for mutein efficacy in vivo.
Assuntos
Linfócitos T CD8-Positivos , Interleucina-2 , Neoplasias , Linfócitos T Reguladores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Interleucina-2/genética , Interleucina-2/imunologia , Melanoma , Camundongos , Mutação , Neoplasias/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Microambiente TumoralRESUMO
Neisseria meningitidis causes meningitis and septicemia. There is no single vaccine against all serogroup B meningococcal (MenB) strains up to now. Their capsular polysaccharide (MenB CPS) bears epitopes both cross-reacting and non-cross-reactive with human polysialic acid. A bactericidal and protective antibody mAb (13D9) recognizing a unique epitope in MenB CPS was used to screen a phage-displayed peptide library. Four peptides, able to bind mAb 13D9 in competition with MenB CPS, were identified. Immunization of mice with the phage-displayed peptides elicited anti-peptide IgG antibodies, mainly IgG(2a) for 3 of the peptides and bactericidal and protective antibody levels for one of them. Peptides specifically targeting the immune response toward epitopes found only in MenB CPS could be considered for a universal vaccine against serogroup B meningococcal strains.
Assuntos
Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Peptídeos/imunologia , Polissacarídeos Bacterianos/imunologia , Animais , Anticorpos Antibacterianos/sangue , Feminino , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Biblioteca de Peptídeos , Ratos , Ensaios de Anticorpos Bactericidas SéricosRESUMO
The number of people with dementia worldwide is estimated at 50 million by 2018 and continues to rise mainly due to increasing aging and population growth. Clinical impact of current interventions remains modest and all efforts aimed at the identification of new therapeutic approaches are therefore critical. Previously, we showed that JM-20, a dihydropyridine-benzodiazepine hybrid molecule, protected memory processes against scopolamine-induced cholinergic dysfunction. In order to gain further insight into the therapeutic potential of JM-20 on cognitive decline and Alzheimer's disease (AD) pathology, here we evaluated its neuroprotective effects after chronic aluminum chloride (AlCl3) administration to rats and assessed possible alterations in several types of episodic memory and associated pathological mechanisms. Oral administration of aluminum to rodents recapitulates several neuropathological alterations and cognitive impairment, being considered a convenient tool for testing the efficacy of new therapies for dementia. We used behavioral tasks to test spatial, emotional- associative and novel object recognition memory, as well as molecular, enzymatic and histological assays to evaluate selected biochemical parameters. Our study revealed that JM-20 prevented memory decline alongside the inhibition of AlCl3 -induced oxidative stress, increased AChE activity, TNF-α and pro-apoptotic proteins (like Bax, caspase-3, and 8) levels. JM-20 also protected against neuronal damage in the hippocampus and prefrontal cortex. Our findings expanded our understanding of the ability of JM-20 to preserve memory in rats under neurotoxic conditions and confirm its potential capacity to counteract cognitive impairment and etiological factors of AD by breaking the progression of key steps associated with neurodegeneration.
Assuntos
Cloreto de Alumínio/toxicidade , Benzodiazepinas/farmacologia , Transtornos da Memória/induzido quimicamente , Memória/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Niacina/análogos & derivados , Cloreto de Alumínio/antagonistas & inibidores , Animais , Hipocampo/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Teste do Labirinto Aquático de Morris/efeitos dos fármacos , Niacina/farmacologia , Teste de Campo Aberto/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos Wistar , Teste de Desempenho do Rota-RodRESUMO
Controlling the global COVID-19 pandemic depends, among other measures, on developing preventive vaccines at an unprecedented pace. Vaccines approved for use and those in development intend to elicit neutralizing antibodies to block viral sites binding to the host's cellular receptors. Virus infection is mediated by the spike glycoprotein trimer on the virion surface via its receptor binding domain (RBD). Antibody response to this domain is an important outcome of immunization and correlates well with viral neutralization. Here, we show that macromolecular constructs with recombinant RBD conjugated to tetanus toxoid (TT) induce a potent immune response in laboratory animals. Some advantages of immunization with RBD-TT conjugates include a predominant IgG immune response due to affinity maturation and long-term specific B-memory cells. These result demonstrate the potential of the conjugate COVID-19 vaccine candidates and enable their advance to clinical evaluation under the name SOBERANA02, paving the way for other antiviral conjugate vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Toxoide Tetânico/química , Vacinas Conjugadas/administração & dosagem , Animais , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinação , Vacinas Conjugadas/imunologiaRESUMO
The development of new immune potentiators for human vaccines is an important and expanding field of research. In the present study, the ability of the capsular polysaccharide from Neisseria meningitidis serogroup A (CPS-A), a mannose-containing carbohydrate, to enhance the antibody production against a co-administered model vaccine antigen, is examined. A protein-meningococcal serogroup C capsular polysaccharide (CPS-C) conjugate was selected as the model antigen for this study. After subcutaneous immunization of Balb/C mice, the conjugate mixed with CPS-A induced higher anti-CPS-C IgG and IgG(2a) antibody levels and higher anti-meningococcal serogroup C bactericidal titers than the conjugate alone or mixed with CPS-C. The immuno-stimulatory properties exhibited by CPS-A and the fact that vaccines based on purified CPS-A has been safely used during decades to fight the serogroup A meningococcal disease, support the proposal to use CPS-A as immune potentiator for human vaccination studies.
Assuntos
Adjuvantes Imunológicos/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/isolamento & purificação , Neisseria meningitidis Sorogrupo A/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Cápsulas Bacterianas/administração & dosagem , Atividade Bactericida do Sangue , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Meningite Meningocócica/imunologia , Meningite Meningocócica/microbiologia , Meningite Meningocócica/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Vacinas Meningocócicas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis Sorogrupo A/química , Polissacarídeos Bacterianos/administração & dosagem , Polissacarídeos Bacterianos/imunologia , Distribuição AleatóriaRESUMO
Selection from a phage display library derived from human Interleukin-2 (IL-2) yielded mutated variants with greatly enhanced display levels of the functional cytokine on filamentous phages. Introduction of a single amino acid replacement selected that way (K35E) increased the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity- the major manufacturability issue limiting IL-2 usefulness up to now. Improvement of secretion was also shown for a panel of IL-2-engineered variants with altered receptor binding properties, including a selective agonist and a super agonist that kept their unique properties. Our findings will improve developability of the growing family of IL-2-derived immunotherapeutic agents and could have a broader impact on the engineering of structurally related four-alpha-helix bundle cytokines.
Assuntos
Substituição de Aminoácidos , Antineoplásicos/farmacologia , Interleucina-2/genética , Receptores Fc/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Sobrevivência Celular/efeitos dos fármacos , Evolução Molecular , Humanos , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Engenharia de Proteínas , Receptores Fc/genéticaRESUMO
The current chapter focuses on the use of filamentous phages to display, modify, and characterize cytokines, which are proteins belonging to a versatile group of essential mediators involved in cell-cell communication. Cytokines exhibit a considerable diversity, both in functions and in structural features underlying their biological effects. A broad variety of cytokines have been successfully displayed on phages, allowing the high-throughput study of their binding properties and biological activities and the discovery of novel therapeutics through directed evolution. The technical singularities and some potential applications of cytokine phage display are illustrated here with the case of Interleukin-2, a prototypic member of the four-alpha-helix bundle cytokine family playing a pivotal role in the immune response and having a long history of therapeutic use.
Assuntos
Interleucina-2/genética , Interleucina-2/imunologia , Biblioteca de Peptídeos , Animais , Linhagem Celular , Humanos , Estrutura Secundária de ProteínaRESUMO
High-dose IL2, first approved in 1992, has been used in the treatment of advanced renal cell carcinoma and melanoma. In these indications, IL2 induces long lasting objective responses in 5% to 20% of patients. However, toxicity and the unexpected expansion of regulatory T cells (Tregs) have limited its practical use and therapeutic impact, respectively. At the Center of Molecular Immunology in Havana, Cuba, a project was launched in 2005 to rationally design IL2 muteins that could be deployed in the therapy of cancer. The basic goal was to uncouple the pleiotropic effect of IL2 on different immune T cells, to obtain a mutein with a therapeutic index that was better than that achieved with wild type (wt) IL2. Using a combination of computational and experimental biology approaches, we predicted and developed two novel IL2 muteins with therapeutic potential. The first, designated no-alpha mutein, is an agonist of IL2R signaling with a reduced ability to expand Treg in vivo. In mice, the no-alpha mutein IL2 has higher antitumor activity and lower toxicity than wt IL2. It represents a potential best-in-class drug that has begun phase I/II clinical trials in solid tumors. The second, designated no-gamma mutein, is an antagonist of IL2R signaling, with some preferential affinity for Tregs. This mutein has antitumor activity in mice that likely derives from its ability to reduce Treg accumulation in vivo. It represents a first-in-class drug that offers a novel strategy to inhibit Treg activity in vivo.
Assuntos
Biologia Computacional/métodos , Modelos Animais de Doenças , Interleucina-2/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Humanos , Interleucina-2/genética , Interleucina-2/imunologia , Mutação , Neoplasias/imunologia , Receptores de Interleucina-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
In this study we compared the following ELISA protocols to measure antibody levels against serogroup C meningococcal polysaccharide: a traditional protocol using poly-L-Lysine mixed with the polysaccharide as coating antigen, a second protocol coating with a mixture of methylated human serum albumin with the C polysaccharide, a modified protocol coating with derivatized polysaccharide and a modification to the last one, specifically without adding ammonium thiocyanate to the sample buffer. Serum bactericidal activity of mouse, monkey and human sera were measured and correlation coefficients were calculated. For all serum types the modified ELISA protocol showed the highest correlation coefficients while the traditional protocol showed the lower ones. We demonstrated that the traditional protocol measures non-specific antibodies to the C polysaccharide, because no differences were detected between pre-immune and post-immune human sera (P>0.05), while the modified protocol detected the highest difference (P<0.01).
Assuntos
Anticorpos Antibacterianos/biossíntese , Ensaio de Imunoadsorção Enzimática/métodos , Meningite Meningocócica/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo C/imunologia , Animais , Anticorpos Antibacterianos/sangue , Cápsulas Bacterianas/imunologia , Chlorocebus aethiops , Feminino , Humanos , Meningite Meningocócica/microbiologia , Meningite Meningocócica/patologia , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos Bacterianos/imunologia , Estatísticas não Paramétricas , Vacinas Conjugadas/imunologiaRESUMO
Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo C/imunologia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Adipatos , Aminação , Animais , Anticorpos Antibacterianos/sangue , Carbodi-Imidas , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Imunoglobulina G/sangue , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/química , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Neisseria meningitidis Sorogrupo C/crescimento & desenvolvimento , Ratos , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
The functional dichotomy of antibodies against interleukin-2 (IL-2) is thought to depend upon recognition of different cytokine epitopes. Beyond functional studies, the only molecular evidence obtained so far located the epitopes recognized by the immunoenhancing antibodies S4B6 and JES6-5H4 within the predicted interface of IL-2 with the α receptor subunit, explaining the preferential stimulation of effector cells displaying only ß and γ receptor chains. A consistent functional map of the epitope bound by the immunoregulatory antibody JES6-1A12 has now been delineated by screening the interactions of phage-displayed antigen variants (with single and multiple mutations) and antigen mimotopes. The target determinant resides in a region between the predicted interfaces with α and ß/γ receptor subunits, supporting the dual inhibitory role of the antibody on both interactions. Binding by JES6-1A12 would thus convert complexed IL-2 into a very weak agonist, reinforcing the advantage of T regulatory cells (displaying the high affinity αßγ heterotrimeric receptor) to capture the cytokine by competition and expand over effector cells, ultimately resulting in the observed strong tolerogenic effect of this antibody. Detailed knowledge of the epitopes recognized by anti-IL-2 antibodies with either immunoenhancing or immunoregulatory properties completes the molecular scenario underlying their use to boost or inhibit immune responses in multiple experimental systems. The expanded functional mapping platform now available could be exploited to study other interactions involving related molecular pairs with the final goal of optimizing cytokine and anti-cytokine therapies.