RESUMEN

Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env-ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env-ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose in vivo and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env-NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression in vitro and immunogenicity at low doses in vivo are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.IMPORTANCEAn effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher in vivo protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials.

Asunto(s)

Ferritinas , VIH-1 , Nanopartículas , Animales , VIH-1/inmunología , VIH-1/genética , Ratones , Ferritinas/inmunología , Ferritinas/genética , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , ARN Mensajero/inmunología , ARN Mensajero/genética , Anticuerpos Anti-VIH/inmunología , Femenino , Anticuerpos Neutralizantes/inmunología , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Ratones Endogámicos BALB C , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Inmunogenicidad Vacunal , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología

RESUMEN

INTRODUCTION: The present study reported a new immunoblot assay, with revelation by R5- or X4-whole free human immunodeficiency virus (HIV) particles or recombinant gp160. MATERIALS AND METHODS: The assay was optimized to identify cell proteins interacting with HIV. Whole cell lysates were prepared from peripheral blood lymphocytes (PBLs), dendritic cells (DC), monocyte-derived macrophage (MDM), and Henrietta Lacks (Hela, wild-type or transfected with DC-specific intracellular adhesion molecule-3-Grabbing Non-Integrin, HeLa) and Human endometrial cells (HEC-1A) lines; HIV particles used were the R5-tropic HIV-1JRCSF and the X4-tropic HIV-1NDK. RESULTS: Experiments with PBL lysates and both viruses demonstrated different bands, including a unique band at 105-117 kDa in addition to nonspecific bands. The 105-117 kDa band migrated at the same level of that observed in controls using total PBL lysate and anti-CD4 mAb for detection and thus likely corresponds to the cluster difference (CD) 4 complex. Blots using lysates of DCs, MDM, HeLa cell line, and HEC-1A cell line allowed identifying several bands that positions were similar to that seen by recombinant gp160 or whole R5- or X4-HIV particles. CONCLUSION: Blot of whole lysates of various HIV target cells is recognized by free HIV particles and allows identifying a wide range of HIV-interacting cell proteins. Such optimized assay could be useful to recognize new cellular HIV attachment proteins.

RESUMEN

The HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a "closed" conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain-the "SOSIP" construct-has many of the relevant properties; it is also particularly suitable for structure determination. Some single-molecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å. Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM. Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env "spike" and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Microscopía por Crioelectrón/métodos , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/inmunología , VIH-1/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Anticuerpos Neutralizantes/química , Células HEK293 , Proteínas gp160 de Envoltorio del VIH/química , Humanos , Unión Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química

RESUMEN

HIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a "closed" to "open" conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design.IMPORTANCE HIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

Asunto(s)

Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Anticuerpos Neutralizantes/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Seropositividad para VIH , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología

RESUMEN

HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , VIH-1/fisiología , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Virión/metabolismo , Virión/fisiología , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo

RESUMEN

BACKGROUND: The envelope protein of lentiviruses are type I transmembrane proteins, and their transmembrane domain contains conserved potentially charged residues. This highly unusual feature would be expected to cause endoplasmic reticulum (ER) localization. The aim of this study was to determine by which means the HIV-1 Env protein is transported to the cell surface although its transmembrane domain contains a conserved arginine residue. RESULTS: We expressed various chimeric proteins and analyzed the influence of their transmembrane domain on their intracellular localization. The transmembrane domain of the HIV-1 Env protein does not cause ER retention. This is not due to the presence of conserved glycine residues, or to the position of the arginine residue, but to the length of the transmembrane domain. A shortened version of the Env transmembrane domain causes arginine-dependent ER targeting. Remarkably, the transmembrane domain of the HIV-1 Env protein, although it does not confer ER retention, interacts efficiently with negatively charged residues in the membrane. CONCLUSION: These results suggest that the intrinsic properties of the HIV-1 Env transmembrane domain allow the protein to escape ER-retention mechanisms, while maintaining its ability to interact with cellular proteins and to influence cellular physiology.

Asunto(s)

VIH-1/metabolismo , Espacio Intracelular/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Membrana Celular , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Lentivirus/metabolismo , Dominios Proteicos , Transporte de Proteínas , Relación Estructura-Actividad

RESUMEN

The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possesses strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env are responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, the consensus C (ConC) clone, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while they had no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identify novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates.IMPORTANCE A better understanding of the mechanisms that determine the wide range of neutralization sensitivity of circulating primary HIV-1 isolates would provide important information about the natural structural and conformational diversity of HIV-1 Env and how this affects the neutralization phenotype. A useful way of studying this is to determine the molecular basis for the unusually high neutralization sensitivities of the limited number of available tier 1a viruses. This study localized the neutralization sensitivity of MW965.26, an extremely sensitive subtype C-derived primary isolate, to two rare substitutions in the C3 and C5 domains and demonstrated that the sequences at these positions differentially affect the presentation of epitopes recognized by different classes of standard and conformation-dependent broadly neutralizing antibodies. These results provide novel insight into how these regions regulate the neutralization phenotype and provide tools for controlling the Env conformation that could have applications both for structural studies and in vaccine design.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , Genes env/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Sustitución de Medicamentos , Epítopos/genética , Epítopos/inmunología , Genes env/genética , Células HEK293 , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Técnicas In Vitro , Mutación , Pruebas de Neutralización , Fenotipo , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética

RESUMEN

INTRODUCTION: HIV viremia is the essential element for progression of an initial HIV infection into AIDS and death. The currently approved management relies primarily on chemotherapy repressing the HIV replication in the infected CD4+ cells, although with severe systemic adverse effects. The problem is that it does not physically eliminate viruses, which then not only keep infecting healthy cells of these patients, but also promote infections of other people. SPECIFIC AIM: An overall objective of our work is biomolecular engineering of virus apheresis tags (VAT) that eliminate viremias without adverse effects. The specific aim of this project was biomolecular engineering of Human Immunodeficiency Virus Apheresis Tags (HIVAT): CD4-Au-Fe3O4, CD4-SiO2-Fe3O4, anti-gp120-Au-Fe3O4, and anti-gp120-SiO2-Fe3O4. HEALTHY DONORS AND PATIENTS: Per the Institutional Review Board's approval and in compliance with Declaration of Helsinki, healthy donors and patients were presented with Patient Bill of Rights and provided Patient Informed Consent, while all the procedures were pursued by the licensed physicians. MATERIALS AND METHODS: CD4, gp120, gp41, gp160, anti-gp120, p24 were transgenomically expressed. Superparamagnetic core-shell particles (SPM-CSP) were synthesized. SPM-CSP were used as the nucleation centers for assembling the expressed molecules upon them to create virus apheresis tags (VAT). VAT were injected into the blood or lymph acquired from the HIV+ and HBV+ patients followed by apheresis at 0.47 - 9.4 T. VAT efficacy in eliminating viremia was determined through immunoblots, NMR and q-RT-PCR. RESULTS: Treatment of blood or lymph of the HIV+ patients' with VAT followed by virus apheresis resulted in rapid elimination of the HIV viremia. Efficacy of apheresis was contingent upon the gravity of viremia versus doses and regimens of VAT. Importantly, administration of VAT also effectively improved levels of non-infected CD4+ lymphocytes. DISCUSSION / CONCLUSIONS: Herein, we present the proof of concept for a new, effective treatment with virus apheresis tags (VAT), specifically Human Immunodeficiency Virus Apheresis Tags (HIVAT), of the HIV+ patients' blood and lymph, which is eliminating the HIV viremia.It can be easily adapted as treatments of viremias perpetrated by other deadly viruses, which we vigorously pursue.

RESUMEN

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

Asunto(s)

Proteínas gp160 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Pliegue de Proteína , Señales de Clasificación de Proteína , Línea Celular , Humanos , Conformación Proteica , Transporte de Proteínas , Proteolisis

RESUMEN

The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160)3, retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.

Asunto(s)

Antígenos/metabolismo , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Antígenos/química , Epítopos , Células HEK293 , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/genética , Humanos , Conformación Proteica

RESUMEN

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Microdominios de Membrana/química , Esfingomielinas/química , Secuencia de Aminoácidos , Expresión Génica , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Interacciones Huésped-Patógeno , Humanos , Fusión de Membrana , Microdominios de Membrana/inmunología , Microdominios de Membrana/virología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Esfingomielinas/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Ensamble de Virus/inmunología , Liberación del Virus/inmunología

RESUMEN

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. IMPORTANCE: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/antagonistas & inhibidores , Proteínas gp160 de Envoltorio del VIH/inmunología , Inmunotoxinas/farmacología , Antígenos CD4/metabolismo , Línea Celular , Epítopos/inmunología , Proteínas gp160 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Pruebas de Neutralización , Unión Proteica , Multimerización de Proteína

RESUMEN

The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS) and is thus responsible for significant morbidity and mortality worldwide. Despite considerable effort, preparation of an effective vaccine for AIDS has been elusive and it has become clear that a fundamental understanding of the relevant antigenic targets on HIV is essential. The Env trimer spike is the only viral antigen present on the surface of the viral particle and it is the target of all broadly neutralizing antibodies isolated to date. Thus, a soluble, homogeneous, and well-defined preparation of Env trimers is an important first step toward biochemical and structural characterization of the antigenic spike. Phospholipid bilayer nanodiscs represent a relatively new technology that can serve as a platform for the assembly of membrane proteins into a native membrane-like environment. Here we describe the preparation and characterization of unprocessed full-length, natively glycoslyated gp160 Env proteins incorporated into nanodiscs (gp160-ND). The particles are soluble and well defined in the absence of detergent, and possess a morphology anticipated of Env incorporated into a lipid ND. Importantly, the gp160-NDs retain CD4 and Env antibody binding characteristics expected of a functional trimer spike and their incorporation into a lipid membrane allows interrogation of epitopes associated with the membrane-proximal ectodomain region of gp41. These studies provide the groundwork for the use of gp160-ND in more detailed biochemical and structural studies that may set the stage for their use in vaccine development.

Asunto(s)

Vacunas contra el SIDA/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , Nanopartículas/química , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Humanos

RESUMEN

Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1-infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.

Asunto(s)

Antirretrovirales/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , VIH-1/inmunología , Inmunoterapia/métodos , Animales , Antirretrovirales/farmacología , Anticuerpos Neutralizantes/farmacología , Cartilla de ADN/genética , ADN Viral/metabolismo , Dependovirus , Quimioterapia Combinada , Humanos , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Carga Viral/efectos de los fármacos

RESUMEN

UNLABELLED: The human immunodeficiency virus type-1 (HIV-1) gp160 (gp120-gp41 complex) trimer envelope (ENV) protein is a potential vaccine candidate for HIV/AIDS. HIV-1 vaccine development has been problematic and charge polarity as well as sequence variation across clades may relate to the difficulties. Further obstacles are caused by sequence variation between blood and brain-derived sequences, since the brain is a separate compartment for HIV-1 infection. We utilize a threedimensional residue measure of solvent exposure, accessible surface area (ASA), which shows that major segments of gp120 and gp41 known structures are solvent exposed across clades. We demonstrate a large percent sequence polarity for solvent exposed residues in gp120 and gp41. The range of sequence polarity varies across clades, blood, and brain from different geographical locations. Regression analysis shows that blood and brain gp120 and gp41 percent sequence polarity range correlate with mean Shannon entropy. These results point to the use of protein modifications to enhance HIV-1 ENV vaccines across multiple clades, blood, and brain. It should be noted that we do not address the issue of protein glycosylation here; however, this is an important issue for vaccine design and development. ABBREVIATIONS: HIV-1 - human immunodeficiency virus type 1, AIDS - acquired immunodeficiency syndrome, ENV - envelope, gp160 - 160,000d glycoprotein, gp120 - 120,000d glycoprotein, gp41 - 41,000d glycoprotein, LANL - Los Alamos National Laboratories, PDB - Protein Data Bank, HVTN - STEP HIV vaccine trial, AA - amino acids, MSA - multiple sequence alignment, ASA - accessible surface area, SNPs- single nucleotide polymorphisms, HAART - Highly Active Antiretroviral Therapy, CCR5 - C-C chemokine receptor type 5, CNS - central nervous system, HIVE - HIV encephalitis, P - polarity, NP - non-polarity, CTL - cytotoxic T lymphocyte, NIAID - National Institute of Allergy and Infectious Diseases.