RESUMEN
After decades of cancer vaccine efforts, there is an imperious necessity for novel ideas that may result in better tumor control in patients. We have proposed the use of a novel Variable Epitope Library (VEL) vaccine strategy, which incorporates an unprecedented number of mutated epitopes to target antigenic variability and break tolerance against tumor-associated antigens. Here, we used an oncofetal antigen/immature laminin receptor protein-derived sequence to generate 9-mer and 43-mer VEL immunogens. 4T1 tumor-bearing mice developed epitope-specific CD8+IFN-γ+ and CD4+IFN-γ+ T cell responses after treatment. Tumor and lung analysis demonstrated that VELs could increase the number of tumor-infiltrating lymphocytes with diverse effector functions while reducing the number of immunosuppressive myeloid-derived suppressor and regulatory T cells. Most importantly, VEL immunogens inhibited tumor growth and metastasis after a single dose. The results presented here are consistent with our previous studies and provide evidence for VEL immunogens' feasibility as promising cancer immunotherapy.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias de la Mama , Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Receptores de Laminina/inmunología , Animales , Vacunas contra el Cáncer/farmacología , Modelos Animales de Enfermedad , Mapeo Epitopo/métodos , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Immune tolerance is the main challenge in the field of cancer vaccines, so modified peptide sequences or naturally occurring mutated versions of cancer-related wild-type (WT) antigens represent a promising pathway. However, the low immunogenicity of mutation-induced neoantigens and, particularly, their incapacity to activate CD8+ T cells are generating doubts on the success of neoantigen-based cancer vaccines in clinical trials. We developed a novel vaccine approach based on a new class of vaccine immunogens, called variable epitope libraries (VELs). We used three regions of survivin (SVN), composed of 40, 49 and 51 amino acids, along with the complete SVN protein to generate the VELs as multiepitope vaccines. BALB/c mice, challenged with the aggressive and highly metastatic 4T1 cell line, were vaccinated in a therapeutic setting. We showed significant tumor growth inhibition and, most importantly, strong suppression of lung metastasis after a single immunization using VEL vaccines. We demonstrated vaccine-induced broad cellular immune responses concomitant with extensive tumor infiltration of T cells, the activation of CD107a+ IFN-γ+ T cells in the spleen and a significant increase in the number of CD3+ CD8+ Ly6C+ effector T cells. In addition, we observed the presence of interferon-γ-, granzyme B- and perforin-producing lymphocytes along with modifications in the amount of CD11b+ Ly6Cint/low Ly6G+ granulocytic myeloid-derived suppressor cells and CD4+ CD25+ FoxP3+ regulatory T cells in the lungs and tumors of mice. In summary, we showed that the VELs represent a potent new class of cancer immunotherapy and propose the application of the VEL vaccine concept as a true alternative to currently available vaccine platforms.
Asunto(s)
Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Células Supresoras de Origen Mieloide/inmunología , Survivin/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Biblioteca de Péptidos , Survivin/genética , Survivin/inmunología , VacunaciónRESUMEN
It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and ß-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.
Asunto(s)
Secuencia Conservada , Lactobacillus acidophilus/enzimología , Almidón/metabolismo , alfa-Amilasas/química , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Glucanos/metabolismo , Mutagénesis , Mutación Puntual , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , alfa-Amilasas/genéticaRESUMEN
The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Proteínas Inhibidoras de la Apoptosis/genética , Neoplasias Mamarias Experimentales/inmunología , Proteínas Represoras/genética , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Células Cultivadas , Epítopos de Linfocito T/genética , Femenino , Tolerancia Inmunológica/inmunología , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/prevención & control , Ratones , Ratones Endogámicos BALB C , Biblioteca de Péptidos , Survivin , Escape del Tumor/inmunología , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Encéfalo/fisiopatología , Línea Celular Tumoral , Humanos , Microtúbulos/metabolismo , Péptidos/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Extracellular and intraneuronal formation of amyloid-beta (Abeta) deposits have been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of Abeta neurotoxicity is not completely understood. Previous studies suggest that binding of Abeta with a number of targets have deleterious effects on cellular functions. It has been shown that Abeta directly interacted with intracellular protein ERAB (endoplasmic reticulum amyloid beta-peptide-binding protein) also known as ABAD (Abeta-binding alcohol dehydrogenase) resulting in mitochondrial dysfunction and cell death. In the present study we have identified another mitochondrial enzyme, ND3 of the human complex I, that binds to Abeta1-42 by the screening of a human brain cDNA library expressed on M13 phage. Our results indicated a strong interaction between Abeta and a phage-displayed 25 amino acid long peptide TTNLPLMVMSSLLLIIILALSLAYE corresponding to C-terminal peptide domain of NADH dehydrogenase, subunit 3 (MTND3) encoded by mitochondrial DNA (mtDNA). This interaction may explain, in part, the inhibition of complex I activity in astrocytes and neurons in the presence of Abeta, described recently. To our knowledge, the present study is the first demonstration of interaction between Abeta and one of the subunits of the human complex I.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Biblioteca de Genes , Pruebas Genéticas/métodos , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Southern Blotting/métodos , Fragmentación del ADN/fisiología , Complejo I de Transporte de Electrón , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Reacción en Cadena de la Polimerasa/métodosRESUMEN
The HIV-1 envelope glycoprotein gp41 contains Cys(X)5Cys motif, which has been shown to elicit a strong antibody response in almost all HIV-1 infected individuals. This disulfide-bonded loop region is conserved in most retroviruses suggesting the existence of an essential function in virus life cycle. In this study, we displayed the peptides comprising 12 amino acids of the immunodominant loop of gp41 on the surface of M13 phage as N-terminal fusions to the minor coat protein pIII and major coat protein pVIII of the phage and demonstrated that cysteine loop containing peptide expressed on phage recognized 62 out of 63 (98.4%) HIV-1 positive samples but not control negative sera while phage bearing linear peptides detected 4-30% of HIV-1-positive sera. The main advantage of phage-based ELISA or other antibody detection-based diagnostic tests of HIV-infection to be used for massive screening in developing countries is the reproducible, simple, rapid and low-cost production of recombinant antigens.
Asunto(s)
Disulfuros/química , Antígenos VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/inmunología , Biblioteca de Péptidos , Unión Competitiva , Disulfuros/inmunología , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/química , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Mercaptoetanol/farmacología , Sustancias Reductoras/farmacología , Sensibilidad y EspecificidadRESUMEN
Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.
Asunto(s)
Mamastrovirus/metabolismo , Sistemas de Lectura Abierta/genética , Poliproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas no Estructurales Virales/metabolismo , Células CACO-2 , Humanos , Mamastrovirus/clasificación , Mamastrovirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serotipificación , Proteínas no Estructurales Virales/genéticaRESUMEN
Human astroviruses are an important cause of gastroenteritis. As part of a molecular epidemiological study carried out in Mexico a human astrovirus isolate, Yuc-8, was adapted to grow in CaCo-2 cells, and its entire genome was sequenced. A 15 amino acid deletion in ORF1a, which has been associated with adaptation of astroviruses to grow in cells other than CaCo-2, was present in Yuc-8. Comparative sequence analysis of the Yuc-8 ORF2 with reported human astrovirus sequences revealed that this isolate belongs to genotype (serotype) 8. Two distinct domains in ORF2 were observed: an amino-terminal domain (residues 1 to 415), with identities higher than 81% among the strains analysed, and a carboxy-terminal domain (residues 416 to 782) with identities between 36 and 60%. Two non-superimposable phylogenetic trees were generated by separate analysis of these two domains, suggesting that a differential selective pressure is exerted along the structural polyprotein.