Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Proc Natl Acad Sci U S A ; 121(22): e2317230121, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38768344

RESUMEN

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.


Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Proteína gp41 de Envoltorio del VIH , VIH-1 , Proteína gp41 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , VIH-1/inmunología , Animales , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Humanos , Ratones , Epítopos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Péptidos/inmunología , Péptidos/química , Femenino , Anticuerpos Monoclonales/inmunología
3.
Protein Sci ; 27(11): 1923-1941, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30144190

RESUMEN

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Animales , Línea Celular , Chlamydia muridarum/química , Chlamydia trachomatis/química , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular/métodos , Cricetulus , Humanos , Peso Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Serogrupo , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Vacunas de Subunidad/química
4.
Hum Vaccin Immunother ; 9(3): 488-96, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23249976

RESUMEN

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.


Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Vacunas de Subunidad/inmunología , Adyuvantes Inmunológicos/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Femenino , Macaca mulatta , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Infecciones Estreptocócicas/inmunología , Vacunas Estreptocócicas/administración & dosificación , Vacunas Estreptocócicas/genética , Vacunas Estreptocócicas/metabolismo , Streptococcus pyogenes/genética , Análisis de Supervivencia , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/genética , Vacunas Conjugadas/inmunología , Vacunas Conjugadas/metabolismo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/metabolismo
5.
J Alzheimers Dis ; 25(4): 655-69, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21483096

RESUMEN

Accumulation of small soluble assemblies of amyloid-ß (Aß)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aß(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aß(42). Modeling suggests that oligomerized Aß(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aß(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aß(42) oligomerization. Using ELISA and AlphaLISA, we found that Aß(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aß(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aß(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aß(42) oligomerization.


Asunto(s)
Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/biosíntesis , Inmunoensayo/métodos , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/biosíntesis , Enfermedad de Alzheimer/inmunología , Animales , Dimerización , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Luz , Microscopía de Fuerza Atómica , Neuronas/metabolismo , Conformación Proteica , Ratas , Reproducibilidad de los Resultados , Dispersión de Radiación
6.
J Biol Chem ; 285(52): 40604-11, 2010 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-20943652

RESUMEN

We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1. We exploited the antigen-binding property of D5 to select complementary small molecules using a high throughput screen of a diverse chemical collection. The resulting small molecule leads were rendered immunogenic by linking them to a carrier protein and were shown to elicit N-heptad repeat-binding antibodies in a fraction of immunized mice. Plasma from HIV-1-infected subjects shown previously to contain broadly neutralizing antibodies was found to contain antibodies capable of binding to haptens represented in the benzylpiperidine leads identified as a result of the high throughput screen, further validating these molecules as vaccine leads. Our results suggest a new paradigm for vaccine discovery using a medicinal chemistry approach to identify lead molecules that, when optimized, could become vaccine candidates for infectious diseases that have been refractory to conventional vaccine development.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Peptidomiméticos/inmunología , Vacunas contra el SIDA/farmacología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/prevención & control , Haptenos/inmunología , Haptenos/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Peptidomiméticos/farmacología
7.
Proc Natl Acad Sci U S A ; 107(31): 13701-6, 2010 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-20615991

RESUMEN

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.


Asunto(s)
Escherichia coli/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Dicroismo Circular , Escherichia coli/genética , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Mutación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína
8.
Proc Natl Acad Sci U S A ; 107(23): 10655-60, 2010 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-20483992

RESUMEN

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Materiales Biomiméticos , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Sueros Inmunes/inmunología , Vacunación , Secuencia de Aminoácidos , Animales , Cobayas , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , VIH-1/aislamiento & purificación , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Conejos
9.
Exp Neurol ; 223(2): 394-400, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19744481

RESUMEN

Amyloid plaque deposition in the brain is a hallmark of Alzheimer's disease, but recent evidence indicates that the disease may be primarily caused by soluble amyloid-beta (1-42) (Abeta) oligomers or Abeta-derived diffusible ligands (ADDLs). ADDLs induce cognitive deficits in animal models and are thought to assemble in vitro by a mechanism apart from plaque formation. To investigate the in vivo relationship of ADDLs and plaques, biotin-labeled ADDLs (bADDLs) or amylin oligomers (bAMs) were injected into the hippocampus of hAPP overexpressing mice. The brains were collected 1 or 5 weeks after the last treatment and were processed for immunohistochemistry. Staining of tissue 1 week post-treatment showed bADDLs had diffused throughout the tissue and incorporated into plaques. Additionally, small deposits of thioflavin S-negative bADDLs were observed. At 5 weeks post-treatment, thioflavin S-positive material continued to accumulate around plaques containing bADDLs. Thioflavin S-positive material also accrued around bADDL deposits, implying that bADDLs were capable of seeding new plaques. In contrast, bAMs cleared from the brain and did not accumulate in plaques. Together, these data indicate that ADDLs are able to contribute to in vivo plaque formation in a peptide-specific manner.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Amiloide/química , Amiloide/genética , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Benzotiazoles , Biotina , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Polipéptido Amiloide de los Islotes Pancreáticos , Ligandos , Masculino , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Tiazoles/metabolismo
10.
Bioconjug Chem ; 19(1): 322-6, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18072716

RESUMEN

Amino acid analysis using direct electrochemical detection was compared with precolumn fluorescent derivatization using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) for evaluation of the degree of covalent coupling of peptides to a carrier-protein complex derived from the bacteria Neisseria meningitidis. AQC derivatization was found to give superior sensitivity compared to electrochemical detection, with less interference from sample components such as carbohydrates or buffer salts. Hydrolysis time and temperature were optimized for maximal recoveries of the marker amino acid 6-aminohexanoic acid (epsilon-Ahx) and the unique amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCHMC), which are generated upon the hydrolysis of the covalent linkage between the peptide and the carrier protein. Quantitation of these amino acids enabled the determination of the ratio of peptide to protein in the conjugate samples.


Asunto(s)
Aminoácidos/análisis , Aminoquinolinas/química , Carbamatos/química , Cromatografía por Intercambio Iónico/métodos , Electroquímica/métodos , Péptidos/análisis , Péptidos/química , Vacunas Conjugadas/química , Ácido Aminocaproico , Proteínas Bacterianas/química , Cromatografía Líquida de Alta Presión , Cisteína/análogos & derivados , Cisteína/análisis , Homocisteína/análogos & derivados , Homocisteína/análisis , Hidrólisis , Neisseria meningitidis/química , Sensibilidad y Especificidad , Temperatura , Factores de Tiempo
11.
Biochemistry ; 45(51): 15157-67, 2006 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-17176037

RESUMEN

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.


Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Soluciones , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/ultraestructura , Animales , Células Cultivadas , Cromatografía en Gel , Ligandos , Microscopía de Fuerza Atómica , Peso Molecular , Neuronas/química , Neuronas/metabolismo , Neuronas/ultraestructura , Fragmentos de Péptidos/toxicidad , Fragmentos de Péptidos/ultraestructura , Unión Proteica , Ratas
12.
Vaccine ; 24(10): 1501-14, 2006 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-16271808

RESUMEN

Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.


Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Codón , Femenino , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...