RESUMEN
Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included.
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Inmunoconjugados , Inmunoconjugados/química , Anticuerpos Monoclonales/químicaRESUMEN
Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.
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Hipercolesterolemia Familiar Homocigótica , Nanopartículas , Animales , Edición Génica/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Hígado/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Lipoproteínas LDL/metabolismoRESUMEN
Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.
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Sistemas CRISPR-Cas , LDL-Colesterol/sangre , Edición Génica , Modelos Animales , Proproteína Convertasa 9/genética , Adenina/metabolismo , Animales , Células Cultivadas , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/enzimología , Mutación con Pérdida de Función , Macaca fascicularis/sangre , Macaca fascicularis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/metabolismo , Factores de TiempoRESUMEN
As one of the leading causes of central vision loss in elderly population, worldwide cases of age-related macular degeneration (AMD) have seen a dramatic increase over the past several years. Treatment regimens for AMD, especially with biological agents, are complicated due to anatomical and physiological barriers, as well as administration of high doses and frequent regimens. Some clinical examples include monthly intravitreal administration of anti-VEGF antibody ranibizumab (Lucentis®) from Genentech and aflibercept (Eylea®) from Regeneron Pharmaceuticals. Long-acting sustained intraocular drug delivery provides promising solutions, such as Vitrasert® from Bausch & Lomb, an intravitreal biodegradable polymeric implant made from poly(D,L-lactic co glycolic acid) (PLGA), and can be used as a guiding reference to formulate sustained delivery systems. In this review, we discuss the anatomy and physiology of the eye, barriers to delivery, pathology of AMD, opportunities for biological therapeutics, and future prospects of intraocular delivery strategies that are in development for treatment of AMD.
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Productos Biológicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Degeneración Macular/tratamiento farmacológico , Animales , Ojo/anatomía & histología , Ojo/efectos de los fármacos , Humanos , Inyecciones Intraoculares , Degeneración Macular/fisiopatología , Fenómenos Fisiológicos Oculares/efectos de los fármacosRESUMEN
The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2-20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.
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Adyuvantes Inmunológicos , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Reacciones Antígeno-Anticuerpo , Células CHO , Cricetinae , Cricetulus , Epítopos/inmunología , Femenino , Glicosilación , Cobayas , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , Antígenos VIH/genética , Antígenos VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/prevención & control , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Polisorbatos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Escualeno/inmunologíaRESUMEN
Adjuvants are necessary to enable vaccine development against a significant number of challenging pathogens for which effective vaccines are not available. We engineered a novel small-molecule immune potentiator, a benzonaphthyridine agonist targeting toll-like receptor 7 (TLR7), as a vaccine adjuvant. TLR7 agonist (TLR7a) was engineered to be adsorbed onto aluminum hydroxide (AlOH), and the resulting AlOH/TLR7a was evaluated as a vaccine adjuvant. AlOH/TLR7a exploits the flexibility of AlOH formulations, has an application in many vaccine candidates, and induced good efficacy and safety profiles against all tested antigens (bacterial- and viral-derived protein antigens, toxoids, glycoconjugates, and so forth) in many animal models, including nonhuman primates. In this article, we describe the outcome of the physicochemical characterization of AlOH/TLR7a. Reverse-phase ultra performance liquid chromatography, confocal microscopy, flow cytometry, zeta potential, and phosphophilicity assays were used as tools to demonstrate the association of TLR7a to AlOH and to characterize this novel formulation. Raman spectroscopy, nuclear magnetic resonance, and mass spectroscopy were also used to investigate the interaction between TLR7a and AlOH (data not shown). This pivotal work paved the way for AlOH/TLR7a to progress into the clinic for evaluation as an adjuvant platform for vaccines against challenging preventable diseases.
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Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , Naftiridinas/química , Receptor Toll-Like 7/agonistas , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Adsorción , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/farmacología , Animales , Humanos , Naftiridinas/administración & dosificación , Naftiridinas/farmacologíaRESUMEN
Adjuvants have a critical role for improving vaccine efficacy against many pathogens, including HIV. Here, using transcriptional RNA profiling and systems serology, we assessed how distinct innate pathways altered HIV-specific antibody responses in nonhuman primates (NHPs) using 8 clinically based adjuvants. NHPs were immunized with a glycoprotein 140 HIV envelope protein (Env) and insoluble aluminum salts (alum), MF59, or adjuvant nanoemulsion (ANE) coformulated with or without Toll-like receptor 4 (TLR4) and 7 agonists. These were compared with Env administered with polyinosinic-polycytidylic acid:poly-L-lysine, carboxymethylcellulose (pIC:LC) or immune-stimulating complexes. Addition of the TLR4 agonist to alum enhanced upregulation of a set of inflammatory genes, whereas the TLR7 agonist suppressed expression of alum-responsive inflammatory genes and enhanced upregulation of antiviral and interferon (IFN) genes. Moreover, coformulation of the TLR4 or 7 agonists with alum boosted Env-binding titers approximately threefold to 10-fold compared with alum alone, but remarkably did not alter gene expression or enhance antibody titers when formulated with ANE. The hierarchy of adjuvant potency was established after the second of 4 immunizations. In terms of antibody durability, antibody titers decreased â¼10-fold after the final immunization and then remained stable after 65 weeks for all adjuvants. Last, Env-specific Fc-domain glycan structures and a series of antibody effector functions were assessed by systems serology. Antiviral/IFN gene signatures correlated with Fc-receptor binding across all adjuvant groups. This study defines the potency and durability of 8 different clinically based adjuvants in NHPs and shows how specific innate pathways can alter qualitative aspects of Env antibody function.
RESUMEN
Although glycoconjugate vaccines are generally very efficacious, there is still a need to improve their efficacy, especially in eliciting a strong primary antibody response. We have recently described a new type of vaccine adjuvant based on a TLR7 agonist adsorbed to alum (Alum-TLR7), which is highly efficacious at enhancing immunogenicity of protein based vaccines. Since no adjuvant has been shown to potentiate the immune response to glycoconjugate vaccines in humans, we investigated if Alum-TLR7 is able to improve immunogenicity of this class of vaccines. We found that in a mouse model Alum-TLR7 greatly improved potency of a CRM197-MenC vaccine increasing anti-MenC antibody titers and serum bactericidal activity (SBA) against MenC compared to alum adjuvanted vaccine, especially with a low dose of antigen and already after a single immunization. Alum-TLR7 also drives antibody response towards Th1 isotypes. This adjuvant was also able to increase immunogenicity of all polysaccharides of a multicomponent glycoconjugate vaccine CRM197-MenACWY. Furthermore, we found that Alum-TLR7 increases anti-polysaccharide immune response even in the presence of a prior immune response against the carrier protein. Finally, we demonstrate that Alum-TLR7 adjuvant effect requires a functional TLR7. Taken together, our data support the use of Alum-TLR7 as adjuvant for glycoconjugate vaccines.
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Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antibacterianos/biosíntesis , Glicoconjugados/administración & dosificación , Meningitis Meningocócica/prevención & control , Vacunas Meningococicas/administración & dosificación , Receptor Toll-Like 7/administración & dosificación , Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Femenino , Glicoconjugados/química , Humanos , Inmunogenicidad Vacunal , Inmunoglobulina G/biosíntesis , Meningitis Meningocócica/inmunología , Meningitis Meningocócica/microbiología , Vacunas Meningococicas/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neisseria meningitidis/efectos de los fármacos , Neisseria meningitidis/inmunología , Receptor Toll-Like 7/química , Vacunación , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/biosíntesisRESUMEN
A panel of researchers working in different areas of adjuvanted vaccines deliberated over the topic, "Gaps in knowledge and prospects for research of adjuvanted vaccines" at, "Enhancing Vaccine Immunity and Value" conference held in July 2014. Several vaccine challenges and applications for new adjuvant technologies were discussed.
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Adyuvantes Inmunológicos/aislamiento & purificación , Adyuvantes Inmunológicos/farmacología , Descubrimiento de Drogas/métodos , Vacunas/inmunología , Vacunas/aislamiento & purificación , Investigación Biomédica/métodos , HumanosRESUMEN
Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIV(AD8) infection and Env protein vaccination with eight different adjuvants. A subset of the SHIV(AD8)-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies.
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Vacunas contra el SIDA/inmunología , Adyuvantes Inmunológicos , Infecciones por VIH/prevención & control , VIH-1/inmunología , Inmunoglobulinas/inmunología , ARN Mensajero/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Secuencia de Bases , Infecciones por VIH/inmunología , Inmunoglobulinas/genética , Infecciones por Lentivirus/inmunología , Infecciones por Lentivirus/prevención & control , Lentivirus de los Primates/inmunología , Estudios Longitudinales , Macaca mulatta , Datos de Secuencia Molecular , ARN Viral/análisis , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Carga ViralRESUMEN
Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.
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Adyuvantes Inmunológicos/farmacología , Diseño de Fármacos , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Disponibilidad BiológicaRESUMEN
As novel vaccine antigens and adjuvants are being tested in humans, understanding of critical quality attributes essential for eliciting optimal vaccine response and vaccine antigen-adjuvant interactions is pivotal for vaccine safety and eliciting 'protective' immune responses. Therefore, the efforts to better characterize and evaluate vaccine antigen and antigen-adjuvant drug products need to begin very early during the discovery and development phase. In this review, we discuss the importance of characterization of physicochemical and functional properties in vaccine antigen, adjuvant and the final antigen-adjuvant drug product and emphasize the greater need for more extensive understanding of vaccine antigen-adjuvant interactions. We highlight the key parameters and quality attributes that are critical to measure during preclinical and clinical testing of the vaccine and discuss in some detail the technologies, and their limitations, used in analyzing the key physicochemical and functional attributes of vaccine antigen and antigen-adjuvant drug product.
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Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/fisiología , Antígenos de Histocompatibilidad Clase II/inmunología , Vacunas/química , Vacunas/inmunología , Animales , Fenómenos Químicos , HumanosRESUMEN
Formulation science is an unappreciated and often overlooked aspect in the field of vaccinology. In this review we highlight key attributes necessary to generate well characterized adjuvant formulations. The relationship between the adjuvant and the antigen impacts the immune responses generated by these complex biopharmaceutical formulations. We will use 5 well established vaccine adjuvant platforms; alum, emulsions, liposomes, PLG, and particulate systems such as ISCOMS in addition to immune stimulatory molecules such as MPL to illustrate that a vaccine formulation is more than a simple mixture of component A and component B. This review identifies the challenges and opportunities of these adjuvant platforms. As antigen and adjuvant formulations increase in complexity having a well characterized robust formulation will be critical to ensuring robust and reproducible results throughout preclinical and clinical studies.
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Adyuvantes Farmacéuticos/uso terapéutico , Vacunas , Adyuvantes Farmacéuticos/química , Animales , Química Farmacéutica/métodos , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , HumanosRESUMEN
The importance of the strength of antigen adsorption by aluminum-containing adjuvants on immunopotentiation was studied using HIV 1 SF162dV2gp140 (gp140), a potential HIV/AIDS antigen. The strengths of adsorption by aluminum hydroxide (AH) adjuvant and aluminum phosphate adjuvant, as measured by the Langmuir adsorptive coefficient, were 1900 and 400 mL/mg, respectively. The strength of adsorption by AH was modified by pretreatment of AH with two different concentrations of potassium dihydrogen phosphate to produce phosphate-treated aluminum hydroxide adjuvants having adsorptive coefficients of 1200 and 800 mL/mg. The four adjuvants were used to prepare vaccines containing either 1 or 10 µg of gp140 per dose. Antibody studies in mice revealed that the presence of an adjuvant increased the immune response in comparison with a solution of gp140 when the dose was 1 µg. Furthermore, the immune response was inversely related to the adsorptive coefficient. In contrast, no significant difference in immunopotentiation was observed between treatments in the presence or absence of an adjuvant when the dose of gp140 was 10 µg. Analysis of the binding of gp140 to CD4 and anti-gp140 monoclonal antibodies by surface plasmon resonance suggests that tight binding induced structural changes in the antigen.
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Vacunas contra el SIDA , Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , VIH-1/inmunología , Inmunidad Humoral , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Adsorción , Animales , Formación de Anticuerpos/inmunología , Composición de Medicamentos , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Resonancia por Plasmón de Superficie , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
This study evaluated the feasibility of using γ-irradiation for preparing sterile poly(lactide-co-glycolide) (PLG) formulations for vaccines. PLG microparticles were prepared by water-in-oil-in-water double-emulsion technique and lyophilized. The vials were γ-irradiated for sterilization process. Antigens from Neisseria meningitidis were adsorbed onto the surface of the particles and were characterized for protein adsorption. Antigens adsorbed onto the surface of the irradiated particles within 30 min. Mice were immunized with these formulations, and vaccine potency was measured as serum bactericidal titers. The γ-irradiated PLG particles resulted in equivalent serum bactericidal titers against a panel of five N. meningitidis strains as the nonirradiated PLG particles. The use of PLG polymers with different molecular weights did not influence the vaccine potency. The PLG particles prepared by γ-irradiation of the lyophilized formulations replace the need for aseptic manufacturing of vaccine formulations. This approach may enable the use of PLG formulations with a variety of antigens and stockpiling for pandemics.
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Antígenos Bacterianos/administración & dosificación , Ácido Láctico/química , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Neisseria meningitidis/inmunología , Ácido Poliglicólico/química , Esterilización/métodos , Adsorción , Animales , Antígenos Bacterianos/inmunología , Liofilización , Rayos gamma , Inmunización , Vacunas Meningococicas/inmunología , Ratones , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Recombinant proteins are increasingly being used as a novel approach for antigens in vaccines. These genetically engineered antigens are poorly immunogenic and require a delivery system and adjuvant to elicit their effect at targeted site of action. A delivery system transports the antigen to site of action and an adjuvant activates the cells via interaction with cell receptors and enhances the potency of the antigen. Micro/nanoparticles made from biodegradable and biocompatible polyesters, polylactide-co-glycolides (PLG), have been extensively used as an adjuvant and delivery system. This chapter discusses the applications of PLG micro/nanoparticles as delivery systems and adjuvant for antigens. PLG microparticles are prepared by a solvent evaporation method while nanoparticles are prepared by solvent displacement method. Synthesis of PLG nanoparticles is simpler in comparison to microparticles and unlike microparticles, it also enables particles to be sterile filtered. In a direct comparison using mouse animal model, our group found that microparticles and nanoparticles exhibited similar immunogenic responses. Materials and methods for synthesis and characterization of micro/nanoparticles with adsorbed antigens are discussed in detail.
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Adyuvantes Inmunológicos/química , Antígenos/química , Química Farmacéutica/métodos , Composición de Medicamentos/métodos , Nanopartículas/química , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos/administración & dosificación , Antígenos/metabolismo , Ratones , Nanopartículas/administración & dosificaciónRESUMEN
Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1beta (IL-1beta) by dendritic cells (DCs). The ability of particulates to promote IL-1beta secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1beta secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1beta production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1beta production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigen-specific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b(+)Gr1(-) cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigen-specific cellular immunity.
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Adyuvantes Inmunológicos/farmacología , Proteínas Portadoras/fisiología , Células Dendríticas/metabolismo , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Animales , Formación de Anticuerpos , Caspasa 1/fisiología , Catepsina B/fisiología , Movimiento Celular , Células Cultivadas , Femenino , Interleucina-1beta/biosíntesis , Ácido Láctico/farmacología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Poliestirenos/farmacología , Receptores Toll-Like/fisiologíaRESUMEN
Oligonucleotides, with specific sequence surrounding CpG motifs, appear to be very effective for the induction of a potent Th1 responses. This molecule represents pathogen-associated molecular patterns (PAMPs) that allows the pathogen recognition receptors (PRRs) present on innate immune cells to recognize them and become activated. PAMPs and related compounds are often labelled as immunopotentiators, allowing a clear distinction between them and particulate delivery systems such as emulsions, liposomes, virus-like particles and microparticles. Microparticles prepared from biodegradable, biocompatible polyesters, and poly (lactide co-glycolide) (PLG). They have been proven to be a good particulate delivery system for the co-delivery of antigens and adjuvants. PLG has been used in humans for many years as a resorbable suture material and controlled-release drug delivery systems. It has been demonstrated that antigen presenting cells (APCs) efficiently uptake the PLG microparticles ( approximately 1 microm) both in vivo and in vitro. After uptake, the PLG subsequently induces an antigen specific CTL response in rodents. Several groups, including our group, have evaluated CpG as an immunopotentiator in various formulations and delivery systems (i.e. emulsions and particulate systems). This review will discuss in detail the work conducted so far with CpG using PLG microparticles as a delivery system. We will also discuss the advantages and enhancement of immune properties of formulating CpG (soluble, adsorbed, and encapsulated forms) with PLG microparticles along with future directions for these microparticles with CpG.
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Adyuvantes Inmunológicos/uso terapéutico , Islas de CpG , Sistemas de Liberación de Medicamentos , Oligodesoxirribonucleótidos/uso terapéutico , Poliglactina 910 , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos/administración & dosificación , Antígenos/inmunología , Biotransformación , Portadores de Fármacos , Composición de Medicamentos , Humanos , Liposomas , Microesferas , Nanopartículas , Nanoestructuras , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , SolubilidadRESUMEN
Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein-based vaccines, applies to immunizations with DNA- or RNA-based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide-coglycolide (PLG)-DNA microparticles encoding HIV-gag (PLG-DNA-gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG-DNA-gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, beta7-deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild-type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN-gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN-gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B-cell responses independent of the mucosal homing receptors alpha4beta7 and alphaEbeta7.
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Técnicas de Transferencia de Gen , Anticuerpos Anti-VIH/biosíntesis , VIH-1/inmunología , Cadenas beta de Integrinas/inmunología , Administración Intranasal , Animales , Virus de la Encefalitis Equina Venezolana/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Inmunidad Mucosa , Inmunización/métodos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Poliésteres , ARN Viral/inmunología , Replicón/inmunología , Vacunas de ADN/inmunología , Vagina/inmunologíaRESUMEN
The objective of this work was to evaluate the potency of the CpG containing oligonucleotide encapsulated within poly(lactide-co-glycolide), and coadministered with antigen adsorbed to poly(lactide-co-glycolide) microparticles (PLG particles). The formulations evaluated include, CpG added in soluble form, CpG adsorbed, and CpG encapsulated. The antigen from Neisseria meningitidis serotype B (Men B) was used in these studies. The immunogenicity of these formulations was evaluated in mice. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of a charged surfactant for the formulations. Neisseria meningitidis B protein was adsorbed to the PLG microparticles, with binding efficiency and initial release measured. CpG was either added in the soluble or adsorbed or encapsulated form based on the type of formulation. The binding efficiency, loading, integrity and initial release of CpG and the antigen were measured from all the formulations. The formulations were then tested in mice for their ability to elicit antibodies, bactericidal activity and T cell responses. Encapsulating CpG within PLG microparticles induced statistically significant higher antibody, bactericidal activity and T cell responses when compared to the traditional method of delivering CpG in the soluble form.