RESUMEN
Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) have the capacity to delay viral rebound when administered to people with HIV-1 (PWH) during anti-retroviral therapy (ART) interruption. To further enhance the performance of bNAbs through their Fc effector functions, in particular NK cell-mediated killing of HIV-1 infected cells, we have produced a panel of glyco-engineered (afucosylated) bNAbs with enhanced affinity for Fc gamma receptor IIIa. These afucosylated anti-HIV-1 bNAbs enhance NK cell activation and degranulation compared to fucosylated counterparts even at low antigen density. NK cells from PWH expressing exhaustion markers PD-1 and TIGIT are activated in a similar fashion by afucosylated bNAbs as NK cell from HIV-1 negative individuals. Killing of HIV-1 infected cells is most effective with afucosylated bNAbs 2G12, N6, PGT151 and PGDM1400, whereas afucosylated PGT121 and non-neutralizing antibody A32 only induce minor NK cell-mediated killing. These data indicate that the approach angle and affinity of Abs influence the capacity to induce antibody-dependent cellular cytotoxicity. Thus, afucosylated bNAbs have the capacity to induce NK cell-mediated killing of infected cells, which warrants further investigation of afucosylated bNAb administration in vivo, aiming for reduction of the viral reservoir and ART free durable control.
Asunto(s)
Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH , Infecciones por VIH , VIH-1 , Células Asesinas Naturales , Humanos , VIH-1/inmunología , Células Asesinas Naturales/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , Anticuerpos Anti-VIH/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Neutralizantes/inmunología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , FucosaRESUMEN
Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses.
Asunto(s)
Seropositividad para VIH , VIH-1 , Animales , Anticuerpos ampliamente neutralizantes , Microscopía por Crioelectrón , Anticuerpos Monoclonales , Proteína gp120 de Envoltorio del VIHRESUMEN
The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.
Asunto(s)
Fiebre de Lassa , Nanopartículas , Cobayas , Conejos , Animales , Virus Lassa/química , Anticuerpos Neutralizantes , Fiebre de Lassa/prevención & control , Glicoproteínas , Vacunas SintéticasRESUMEN
Although advances in antiretroviral therapy (ART) have significantly improved the life expectancy of people living with HIV-1 (PLWH) by suppressing HIV-1 replication, a cure for HIV/AIDS remains elusive. Recent findings of the emergence of drug resistance against various ART have resulted in an increased number of treatment failures, thus the development of novel strategies for HIV-1 cure is of immediate need. Antibody-based therapy is a well-established tool in the treatment of various diseases and the engineering of new antibody derivatives is expanding the realms of its application. An antibody-based carrier of anti-HIV-1 molecules, or antibody conjugates (ACs), could address the limitations of current HIV-1 ART by decreasing possible off-target effects, reduce toxicity, increasing the therapeutic index, and lowering production costs. Broadly neutralizing antibodies (bNAbs) with exceptional breadth and potency against HIV-1 are currently being explored to prevent or treat HIV-1 infection in the clinic. Moreover, bNAbs can be engineered to deliver cytotoxic or immune regulating molecules as ACs, further increasing its therapeutic potential for HIV-1 cure. ACs are currently an important component of anticancer treatment with several FDA-approved constructs, however, to date, no ACs are approved to treat viral infections. This review aims to outline the development of AC for HIV-1 cure, examine the variety of carriers and payloads used, and discuss the potential of ACs in the current HIV-1 cure landscape.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , VIH-1/inmunología , Inmunoconjugados/uso terapéutico , Anticuerpos ampliamente neutralizantes/uso terapéutico , Anticuerpos Anti-VIH/uso terapéutico , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Inmunoconjugados/efectos adversos , Inmunoconjugados/inmunología , Receptores CCR5/inmunología , Latencia del VirusRESUMEN
Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.
Asunto(s)
Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Evolución Molecular Dirigida/métodos , Anticuerpos Anti-VIH/inmunología , Inmunogenicidad Vacunal , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/química , Regiones Determinantes de Complementariedad/inmunología , Modelos Animales de Enfermedad , Células HEK293 , Anticuerpos Anti-VIH/química , Humanos , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos B/inmunologíaRESUMEN
The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within â¼24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos ampliamente neutralizantes/metabolismo , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH/inmunología , VIH-1/fisiología , Vacunas contra el SIDA/genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/genética , Afinidad de Anticuerpos , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/genética , Antígenos CD4/metabolismo , Regiones Determinantes de Complementariedad/genética , Anticuerpos Anti-VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
Broadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.
Asunto(s)
Vacunas contra el SIDA/metabolismo , Anticuerpos Neutralizantes/metabolismo , Evolución Molecular , Anticuerpos Anti-VIH/metabolismo , VIH-1/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Sitios de Unión de Anticuerpos , Microscopía por Crioelectrón , Epítopos/química , Epítopos/inmunología , Glicosilación , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Humanos , Simulación del Acoplamiento Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.
Asunto(s)
Anticuerpos Neutralizantes/fisiología , Linaje de la Célula , Anticuerpos Anti-VIH/fisiología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/química , Regiones Determinantes de Complementariedad , Anticuerpos Anti-VIH/química , HumanosRESUMEN
We describe a general method to synthesize the iminium tetrahydrothiophene embedded in the dimeric Nuphar alkaloids. In contrast to prior studies, the sulfur atom of the thiaspirane pharmacophore is shown to be electrophilic. This α-thioether reacts with thiophenol or glutathione at ambient temperature to cleave the C-S bond and form a disulfide. Rates of conversion are proportional to the corresponding ammonium ion pK a and exhibit half-lives less than 5 h at a 5 mM concentration of thiol. A simple thiophane analogue of the Nuphar dimers causes apoptosis at single-digit micromolar concentration and labels reactive cysteines at similar levels as the unsaturated iminium "warhead". Our experiments combined with prior observations suggest the sulfur of the Nuphar dimers can react as an electrophile in cellular environments and that sulfur-triggered retrodimerization can occur in the cell.
RESUMEN
A nitrosopurine ene reaction easily assembles the asmarine pharmacophore and transmits remote stereochemistry to the diazepine-purine hetereocycle. This reaction generates potent cytotoxins which exceed the potency of asmarineâ A (1.2â µM IC50) and supersede the metabolites as useful leads for biological discovery.
Asunto(s)
Diterpenos/química , Purinas/química , Azepinas/química , Supervivencia Celular/efectos de los fármacos , Citotoxinas/síntesis química , Citotoxinas/química , Citotoxinas/toxicidad , Diterpenos/síntesis química , Diterpenos/toxicidad , Células HT29 , Humanos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Caspases are fundamental to many essential biological processes, including apoptosis, differentiation, and inflammation. Unregulated caspase activity is also implicated in the development and progression of several diseases, such as cancer, neurodegenerative disorders, and sepsis. Unfortunately, it is difficult to determine exactly which caspase(s) of the 11 isoforms that humans express is responsible for specific biological functions. This lack of resolution is primarily due to highly homologous active sites and overlapping substrates. Currently available peptide-based inhibitors and probes are based on specificity garnered from peptide substrate libraries. For example, the canonical tetrapeptide LETD was discovered as the canonical sequence that is optimally recognized by caspase-8; however, LETD-based inhibitors and substrates promiscuously bind to other isoforms with equal affinity, including caspases-3, -6, and -9. In order to mitigate this problem, we report the identification of a new series of compounds that are >100-fold selective for inhibiting the initiator caspases-8 and -9 over the executioner caspases-3, -6, and -7.
Asunto(s)
Aminoácidos/química , Inhibidores de Caspasas/farmacología , Caspasas/química , Fragmentos de Péptidos/farmacología , Inhibidores de Caspasas/química , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Biblioteca de Péptidos , Especificidad por SustratoRESUMEN
Wake up, protein! Small molecules that directly activate proteins are rare and their discovery opens new avenues for the development of drugs and chemical tools to probe the functions and mechanisms of protein targets. To address the one-sided dichotomy between enzyme inhibition and activation, we describe a series of procaspase activators as chemical tools in the study of caspase biology.