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1.
Hepatology ; 72(5): 1528-1540, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32770836

RESUMEN

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.


Asunto(s)
Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T/uso terapéutico , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/efectos de los fármacos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Complejo CD3/antagonistas & inhibidores , Línea Celular Tumoral , Epítopos/inmunología , Antígeno HLA-A2/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Activación de Linfocitos/efectos de los fármacos , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T/inmunología
2.
Proc Natl Acad Sci U S A ; 111(51): 18207-12, 2014 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-25489108

RESUMEN

During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates.


Asunto(s)
Fármacos Anti-VIH/farmacología , Proteína gp41 de Envoltorio del VIH/química , Imitación Molecular , Fenómenos Biofísicos , Cristalografía por Rayos X , Escherichia coli/genética , Proteína gp41 de Envoltorio del VIH/genética , Modelos Moleculares
3.
Biophys J ; 111(4): 700-709, 2016 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-27558714

RESUMEN

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.


Asunto(s)
Fenómenos Químicos , Proteína gp41 de Envoltorio del VIH/química , Secuencia de Aminoácidos , Proteína gp41 de Envoltorio del VIH/genética , Modelos Moleculares , Mutación , Dominios Proteicos , Solubilidad
4.
J Biol Chem ; 289(2): 594-9, 2014 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-24302742

RESUMEN

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Epítopos/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Termodinámica , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos/inmunología , Unión Competitiva/inmunología , Calorimetría/métodos , Epítopos/química , Epítopos/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Datos de Secuencia Molecular , Unión Proteica/inmunología
5.
ACS Chem Biol ; 17(10): 2753-2768, 2022 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-36098557

RESUMEN

TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1ß), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33ß, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33ß, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31-27K18Ac, and bind preferentially to H31-27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.


Asunto(s)
Histonas , Factores de Transcripción , Factores de Transcripción/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Lisina/metabolismo , Péptido T/metabolismo , Ligandos , Proteínas de Unión al ADN/metabolismo , Ubiquitinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
6.
Nat Commun ; 10(1): 4521, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31586061

RESUMEN

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.


Asunto(s)
Anticuerpos/química , Dominios PDZ , Péptidos/química , Ingeniería de Proteínas , Mapas de Interacción de Proteínas/efectos de los fármacos , Animales , Anticuerpos/farmacología , Células COS , Chlorocebus aethiops , Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Homólogo 4 de la Proteína Discs Large/metabolismo , Diseño de Fármacos , Mapeo Epitopo , Modelos Moleculares , Biblioteca de Péptidos , Péptidos/farmacología , Unión Proteica , Proteínas Recombinantes/metabolismo
7.
Front Immunol ; 8: 63, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28203239

RESUMEN

Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3 months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission.

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