RESUMEN
The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.
Asunto(s)
VIH-1 , Virión , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , VIH-1/genética , VIH-1/fisiología , VIH-1/inmunología , Virión/metabolismo , Células HEK293 , Anticuerpos Anti-VIH/inmunología , Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/virologíaRESUMEN
HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.
Asunto(s)
Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Virión/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructura , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/ultraestructura , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Secuencia de Aminoácidos , Disulfuros/farmacología , Epítopos/química , Células HEK293 , Proteína gp41 de Envoltorio del VIH/química , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Modelos Moleculares , Pruebas de Neutralización , Péptidos/química , Polisacáridos/química , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Background: Persistence of HIV reservoir even in suppressive ART is the key obstacle in HIV-1 cure. We evaluated the ability of HIV-1 C Env to reactivate the latently infected resting memory CD4 cells and the ability of polyclonal HIV antibodies mediating ADCC to lyse the reactivated targets. Methodology: HIV-1 antibodies from 25 HIV infected individuals (14 ADCC responders and 11 non-responders) were tested against the Env-C reactivated primary cells; CD4+ and CD4+CD45RO+ memory T cells in the presence of autologous or heterologous effector cells using multicolor flow cytometry. The frequencies of p24+ve target cells were measured to determine the reactivation and antibody mediated lysis. Results: Increase in the frequency of p24 expressing cells (P < 0.01 in all cases) after Env-C stimulation of target cells indicated reactivation. When these reactivated targets were mixed with effector cells and HIV-1 antibodies, the frequencies of p24 expressing targets were decreased significantly when the ADCC mediating antibodies (P < 0.01 in all cases) were added but not when the antibodies from ADCC non-responders or HIV negative individuals were added. In parallel, the NK cell activation was also increased only when ADCC mediating antibodies were added. Conclusion: The study showed that the HIV-1 Env could act as latency reversal agent (LRA), and only ADCC mediating antibodies could lyse the reactivated HIV reservoirs. The short stimulation cycle used in this study could be useful in testing LRAs as well as immune mediated lysis of reactivated reservoirs. The observations have further implication in designing antibody mediated immunotherapy for eradication of latent HIV reservoir.
Asunto(s)
Fármacos Anti-VIH/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/inmunología , Latencia del Virus/efectos de los fármacos , Latencia del Virus/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Adulto , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Femenino , Citometría de Flujo , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/genética , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Modelos Biológicos , Provirus/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Despite the success of antiretroviral therapy in suppressing HIV to an undetectable level in the blood and improving patients' quality of life, HIV persists in antiretroviral therapy-treated patients and threatens their lives. Anti-HIV chimeric antigen receptor (CAR) T cells could offer a cure by recognizing and killing virus-producing cells in an Env-specific manner. In this review, the authors summarize several important aspects of the development of anti-HIV CAR T cells, with a special focus on the evolution of CAR design for enhanced potency and targeting specificity, and also outline the challenges that still need to be addressed to take anti-HIV CAR T cells from a hopeful approach to a real HIV cure.
Asunto(s)
Desarrollo de Medicamentos , Infecciones por VIH/inmunología , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Antígenos CD4/inmunología , Desarrollo de Medicamentos/tendencias , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Evasión Inmune/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos/química , Receptores Quiméricos de Antígenos/uso terapéutico , Linfocitos T/trasplante , Linfocitos T/virología , Latencia del Virus/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The envelope protein (Env) is the only surface protein of the human immunodeficiency virus (HIV) and as such the exclusive target for protective antibody responses. Experimental evidences from mouse models suggest a modulating property of Env to steer antibody class switching towards the less effective antibody subclass IgG1 accompanied with strong TH2 helper responses. By simple physical linkage we were able to imprint this bias, exemplified by a low IgG2a/IgG1 ratio of antigen-specific antibodies, onto an unrelated antigen, namely the HIV capsid protein p24. Here, our results indicate the glycan moiety of Env as the responsible immune modulating activity. Firstly, in Card9-/- mice lacking specific C-Type lectin responsiveness, DNA immunization significantly increased the IgG2a/IgG1 ratio for the Env-specific antibodies while the antibody response against the F-protein of the respiratory syncytial virus (RSV) serving as control antigen remained unchanged. Secondly, sequential shortening of the Env encoding sequence revealed the C2V3 domain as responsible for the strong IgG1 responses and TH2 cytokine production. Removing all potential N-glycosylation sites from the C2V3 domain by site-specific mutagenesis reversed the vaccine-induced immune response towards a Th1-dominated T-cell response and a balanced IgG2a/IgG1 ratio. Accordingly, the stretch of oligomannose glycans in the C2V3 domain of Env might mediate a specific uptake and/or signaling modus in antigen presenting cells by involving interaction with an as yet unknown C-type lectin receptor. Our results contribute to a deeper understanding of the impact of Env glycosylation on HIV antigen-specific immune responses, which will further support HIV vaccine development.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Anti-VIH/inmunología , Inmunoglobulina G/inmunología , Vacunas de ADN/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Glicosilación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Th2/inmunología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
Following HIV infection, most people make antibodies to gp120 and gp41, yet only a few make broadly neutralizing antibodies that target key antigenic sites on the envelope glycoproteins. The induction of broadly neutralizing antibodies by immunization remains a major challenge of HIV vaccine research. Difficulties include: variable protein sequence, epitopes that depend on the native conformation, glycosylation that conceals key antigenic determinants, and the assembly of Env trimers that mimic viral spikes. In addition, more potent immunogens may be needed to initiate the response of germline antibody precursors and drive B cell maturation toward antibodies with broad neutralizing activity. We have expressed HIV Env glycoproteins by incorporation into live attenuated rubella viral vectors. The rubella vaccine strain RA27/3 has demonstrated its safety and potency in millions of children. As a vector, it has elicited potent and durable immune responses in macaques to SIV Gag vaccine inserts. We now find that rubella/env vectors can stably express Env core derived glycoproteins ranging in size up to 363 amino acids from HIV clade C strain 426c. The expressed Env glycoproteins bind broadly neutralizing antibodies that target the native CD4 binding site. The vectors grew well in rhesus macaques, and they elicited a vaccine "take" in all animals, as measured by anti-rubella antibodies. By themselves, the vectors elicited modest antibody titers to the Env insert. But the combination of rubella/env prime followed by a homologous protein boost gave a strong response. Neutralizing antibodies appeared gradually after multiple vaccine doses. The vectors will be useful for testing new vaccine inserts and immunization strategies under optimized conditions of vector growth and protein expression.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/prevención & control , Virus de la Rubéola , Animales , Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/inmunología , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/inmunología , VIH-1 , Inmunización Secundaria , Macaca mulatta , Proteínas Recombinantes/inmunología , Vacunas Atenuadas/inmunologíaRESUMEN
INTRODUCTION: Polymorphism in vaccine antigens poses major challenges to vaccinologists. The Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) poses such a challenge. We found that immunization with a mixture of three variants yielded functional antibody levels to all variants comparable to levels induced by monovalent immunization. The mechanism behind the observed broadening was shown to be an increase in the fraction of cross-reactive antibodies, most likely because strain-specific epitopes are present at lower frequency relative to conserved epitopes. Areas covered: We hereby introduce the Epitope Dilution Phenomenon (EDiP) as a practical strategy for the induction of broad, cross-variant antibody responses against polymorphic antigens and discuss the utility and applicability of this phenomenon for the development of vaccines against polymorphic antigens of pathogens like Influenza, HIV, Dengue and Plasmodium. Expert commentary: EDiP can be used to broaden antibody responses by immunizing with a mixture of at least 3 antigenic variants, where the variants included can differ, yet yield broadened responses.
Asunto(s)
Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Variación Antigénica , Antígenos de Protozoos/inmunología , Epítopos , Humanos , Inmunización , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas Protozoarias/inmunologíaRESUMEN
Infection with HIV or SIV often elicits a potent immune response to viral antigens. This includes T cells and antibodies specific for Gag and Env antigens. In contrast, when given as a vaccine, the same antigens have been weak immunogens, unable to elicit antibodies with comparable titer, durability, or neutralizing activity. We have used the live attenuated rubella vaccine strain RA27/3 as a viral vector to express HIV and SIV antigens. By mimicking an HIV infection, these vectors could elicit stronger and more durable immunity to HIV antigens. The vectors are based on the licensed rubella vaccine strain, which has demonstrated safety and potency in millions of children. One or two doses protect for life against rubella infection. The question was whether rubella vectors could similarly enhance the immunogenicity of a foreign vaccine insert. We have previously reported that rubella vectors can express small protein antigens in vitro and in vivo, where they elicit a strong immune response to the vaccine insert. The vectors have now expressed larger vaccine inserts that include epitope-rich fragments of the Gag matrix and capsid proteins (aa 41-211) or the complete p27 capsid protein with p2 (aa 136-381). These vectors have elicited a robust and durable immune response to Gag in rhesus macaques. This size range also encompasses the engineered outer domain (eOD) of HIV envelope gp120 (172 amino acids). The rubella/eOD-GT6 and GT8 vectors stably expressed glycoproteins that bind germline precursors and mature forms of VRC01-class broadly neutralizing antibodies. These vectors potentially could be used as part of a sequential immunization strategy to initiate the production of broadly neutralizing antibodies.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Productos del Gen gag/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Virus de la Rubéola/genética , Virus de la Inmunodeficiencia de los Simios/genética , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Antivirales/inmunología , Productos del Gen gag/genética , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Inmunización , Inmunogenicidad Vacunal , Macaca mulatta , Vacuna contra la Rubéola/genética , Vacuna contra la Rubéola/inmunología , Virus de la Rubéola/inmunología , Vacunas contra el SIDAS/genética , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
Vaccines that protect against viral infections generally induce neutralizing antibodies. When vaccines are evaluated, the need arises to assess the affinity maturation of the antibody responses. Binding titers of polyclonal sera depend not only on the affinities of the constituent antibodies but also on their individual concentrations, which are difficult to ascertain. Therefore an assay based on chaotrope disruption of antibody-antigen complexes was designed for measuring binding strength. This assay works well with many viral antigens but gives differential results depending on the conformational dependence of epitopes on complex antigens such as the envelope glycoprotein of HIV-1. Kinetic binding assays might offer alternatives, since they can measure average off-rate constants for polyclonal antibodies in a serum. Here, potentials and fallacies of these techniques are discussed.