RESUMEN
Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC50) and ibalizumab sensitivity by IC50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to â¼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab.
Asunto(s)
Fármacos Anti-VIH , Farmacorresistencia Viral , Proteína gp120 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Fármacos Anti-VIH/farmacología , Organofosfatos/farmacología , Sitios de Unión , Concentración 50 Inhibidora , Anticuerpos Monoclonales , PiperazinasRESUMEN
The presence of genetic mutations in HIV poses a significant challenge, potentially leading to antiretroviral resistance and hampering therapeutic development. The Brazilian population has presented variations in the HIV envelope V3 loop gene, especially the GWGR motif. This motif has been linked to reduced transmission potential and slower CD4+ T cell decline. This study aimed to assess clinical outcomes in patients with HIV-1 infected with strains containing the GWGR motif compared with those without it during long-term cART. A cohort of 295 patients with HIV was examined for the GWGR motif presence in the V3 loop. A total of 58 samples showed the GWGR signature, while 237 had other signatures. Multifactorial analyses showed no significant differences in demographic characteristics, CD4+ cell count, AIDS progression, or mortality between GWGR carriers and others. However, the mean interval between the first positive HIV test and the initial AIDS-defining event was more than two times longer for women carrying the GWGR signature (p = 0.0231). We emphasize the positive impact of cART on HIV/AIDS treatment, including viral suppression, CD4+ cell preservation, and immune function maintenance. Although no significant differences were found during cART, residual outcomes reflecting adherence challenges were observed between diagnosis and the first AIDS-defining event. The previously described outcomes, highlighting statistically significant differences between individuals carrying the GPGR motif compared with those with the Brazilian GWGR motif, may be directly linked to the natural progression of infection before advancements in cART. Presently, these physicochemical aspects may no longer hold the same relevance.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Femenino , VIH-1/genética , VIH-1/efectos de los fármacos , Masculino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Adulto , Recuento de Linfocito CD4 , Fármacos Anti-VIH/uso terapéutico , Persona de Mediana Edad , Resultado del Tratamiento , Secuencias de Aminoácidos , Carga Viral , Proteína gp120 de Envoltorio del VIH/genética , Estudios de Cohortes , Brasil , Terapia Antirretroviral Altamente Activa , Progresión de la Enfermedad , MutaciónRESUMEN
Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. Fold-change in half-maximal inhibitory concentration (IC50) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC50 and temsavir binding affinity (r = 0.7332; P = 0.0246) as well as temsavir binding on-rate (r = -0.8940; P = 0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations.
Asunto(s)
Fármacos Anti-VIH , Antígenos CD4 , Proteína gp120 de Envoltorio del VIH , VIH-1 , Unión Proteica , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/química , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Fármacos Anti-VIH/farmacología , Antígenos CD4/metabolismo , Antígenos CD4/genética , Sustitución de Aminoácidos , Polimorfismo Genético , Farmacorresistencia Viral , Concentración 50 Inhibidora , CinéticaRESUMEN
Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infections. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoires is still lacking. Here, we develop a straightforward computational method for the Rapid Automatic Identification of bNAbs (RAIN) based on machine learning methods. In contrast to other approaches, which use one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for the accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained and sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of distinct HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using an in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires.
Asunto(s)
Anticuerpos Neutralizantes , Microscopía por Crioelectrón , Anticuerpos Anti-VIH , Infecciones por VIH , VIH-1 , Aprendizaje Automático , VIH-1/inmunología , Humanos , Anticuerpos Anti-VIH/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Antígenos CD4/metabolismo , Antígenos CD4/inmunología , Secuencia de Aminoácidos , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/químicaRESUMEN
The HIV-1 envelope glycoprotein (Env) plays crucial role in viral infection by facilitating viral attachment to host cells and inducing fusion of the virus with the host cell membrane. This fusion allows the HIV-1 viral genome to enter the target cell then triggering various stages of the viral life cycle. The native Env directly interacts with the main receptor CD4 and the co-receptor (CCR5 or CXCR4) in human cell membrane then induces membrane fusion. The elucidation of the structure of Env with CD4 and co-receptors in different HIV-1 subtypes is essential for the understanding of the mechanism of virus entry. Here we report the Cryo-EM structure of the CD4-bound HIV-1 heterotrimeric Env from Asia prevalent CRF07_BC CH119 strain. In this structure, the binding of three CD4 molecules with Env induced extensively conformational changes in gp120, resulting in the transformation of the Env from close state to intermediate open state. Additionally, the conformational shift of V1/V2 loops of the heterotrimeric Env allosterically expose the V3 loop and promoting the further interactions with co-receptor CCR5 or CXCR4. These findings not only illustrate the structural complexity and plasticity of HIV-1 Env but also give new insights how the biological trimeric Env initialize the immune recognition and membrane fusion.
Asunto(s)
Antígenos CD4 , Proteína gp120 de Envoltorio del VIH , VIH-1 , VIH-1/metabolismo , Humanos , Antígenos CD4/metabolismo , Antígenos CD4/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Microscopía por Crioelectrón , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Receptores CCR5/metabolismo , Receptores CCR5/química , Unión Proteica , Modelos Moleculares , Conformación Proteica , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Multimerización de Proteína , Receptores CXCR4/metabolismo , Receptores CXCR4/química , AsiaRESUMEN
BACKGROUND: An effective vaccine is required to end the HIV pandemic. We evaluated the safety and immunogenicity of a DNA (DNA-HIV-PT123) vaccine with low- or high-dose bivalent (TV1.C and 1086.C glycoprotein 120) subtype C envelope protein combinations, adjuvanted with MF59 or AS01B. METHODS: HIV Vaccine Trials Network (HVTN)108 was a randomized, placebo-controlled, double-blind, phase 1/2a trial conducted in the United States and South Africa. HIV-negative adults were randomly assigned to 1 of 7 intervention arms or placebo to assess DNA prime with DNA/protein/adjuvant boosts, DNA/protein/adjuvant co-administration, and low-dose protein/adjuvant regimens. HVTN111 trial participants who received an identical regimen were also included. Outcomes included safety and immunogenicity 2 weeks and 6 months after final vaccination. RESULTS: From June 2016 to July 2018, 400 participants were enrolled (N = 334 HVTN108, N = 66 HVTN111); 370 received vaccine and 30 received placebo. There were 48 grade 3 and 3 grade 4 reactogenicity events among 39/400 (9.8%) participants, and 32 mild/moderate-related adverse events in 23/400 (5.8%) participants. All intervention groups demonstrated high IgG response rates (>89%) and high magnitudes to HIV-1 Env gp120 and gp140 proteins; response rates for AS01B-adjuvanted groups approached 100%. V1V2 IgG magnitude, Fc-mediated functions, IgG3 Env response rates, and CD4+ T-cell response magnitudes and rates were higher in the AS01B-adjuvanted groups. The AS01B-adjuvanted low-dose protein elicited greater IgG responses than the higher protein dose. CONCLUSIONS: The vaccine regimens were generally well tolerated. Co-administration of DNA with AS01B-adjuvanted bivalent Env gp120 elicited the strongest humoral responses; AS01B-adjuvanted regimens elicited stronger CD4+ T-cell responses, justifying further evaluation.ClinicalTrials.gov registration: NCT02915016, registered 26 September 2016.
Asunto(s)
Vacunas contra el SIDA , Adyuvantes Inmunológicos , Anticuerpos Anti-VIH , Proteína gp120 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , Polisorbatos , Escualeno , Vacunas de ADN , Humanos , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/efectos adversos , Vacunas de ADN/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/efectos adversos , Femenino , Masculino , Adulto , Escualeno/administración & dosificación , Polisorbatos/administración & dosificación , Proteína gp120 de Envoltorio del VIH/inmunología , Adyuvantes Inmunológicos/administración & dosificación , VIH-1/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Anticuerpos Anti-VIH/sangre , Método Doble Ciego , Persona de Mediana Edad , Adulto Joven , Adyuvantes de Vacunas/administración & dosificación , Sudáfrica , Inmunogenicidad Vacunal , Adolescente , Estados UnidosRESUMEN
An important approach to stopping the AIDS epidemic is the development of a vaccine that elicits antibodies that block virus capture, the initial interactions of HIV-1 with the target cells, and replication. We utilized a previously developed qRT-PCR-based assay to examine the effects of broadly neutralizing antibodies (bNAbs), plasma from vaccine trials, and monoclonal antibodies (mAbs) on virus capture and replication. A panel of bNAbs inhibited primary HIV-1 replication in PBMCs but not virus capture. Plasma from RV144 and RV305 trial vaccinees demonstrated inhibition of virus capture with the HIV-1 subtype prevalent in Thailand. Several RV305 derived V2-specific mAbs inhibited virus replication. One of these RV305 derived V2-specific mAbs inhibited both virus capture and replication, demonstrating that it is possible to elicit antibodies by vaccination that inhibit virus capture and replication. Induction of a combination of such antibodies may be the key to protection from HIV-1 acquisition.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , VIH-1 , Replicación Viral , VIH-1/inmunología , Humanos , Anticuerpos Anti-VIH/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Monoclonales/inmunología , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Vacunas contra el SIDA/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Anticuerpos ampliamente neutralizantes/inmunologíaRESUMEN
Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment.
Asunto(s)
Escherichia coli , Proteína gp120 de Envoltorio del VIH , Péptidos , Unión Proteica , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Simulación de Dinámica Molecular , Humanos , Secuencia de Aminoácidos , Diseño de FármacosRESUMEN
BACKGROUND: An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. METHODS: HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. FINDINGS: Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. INTERPRETATION: Both vaccine regimens were safe, warranting evaluation in larger trials. FUNDING: US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos Anti-VIH , Infecciones por VIH , VIH-1 , Vacunas de ADN , Humanos , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/efectos adversos , Adulto , Masculino , Femenino , Método Doble Ciego , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas de ADN/efectos adversos , Infecciones por VIH/prevención & control , Infecciones por VIH/inmunología , Persona de Mediana Edad , Adulto Joven , Anticuerpos Anti-VIH/sangre , Adolescente , VIH-1/inmunología , Estados Unidos , Inmunización Secundaria , Inmunogenicidad Vacunal , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Anticuerpos Neutralizantes/sangreRESUMEN
Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos ampliamente neutralizantes , Centro Germinal , Anticuerpos Anti-VIH , VIH-1 , Inmunización Secundaria , Nanopartículas , Vacunas de ARNm , Animales , Humanos , Ratones , Vacunas contra el SIDA/inmunología , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Reacciones Cruzadas , Técnicas de Sustitución del Gen , Centro Germinal/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , VIH-1/genética , Liposomas , Células B de Memoria/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/genética , Hipermutación Somática de Inmunoglobulina , Vacunas de ARNm/inmunología , Femenino , Ratones Endogámicos C57BLRESUMEN
The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Antígenos CD4 , Anticuerpos Anti-VIH , VIH-1 , Humanos , Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Anticuerpos Anti-VIH/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Vacunas de ADN/inmunología , Anticuerpos Monoclonales/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Microscopía por Crioelectrón , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Sitios de Unión , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/químicaRESUMEN
OBJECTIVE: This study aims to investigate the functions and mechanistic pathways of Astrocyte Elevated Gene-1 (AEG-1) in the disruption of the blood-retinal barrier (BRB) caused by the HIV-1 envelope glycoprotein gp120. DESIGN: We utilized ARPE-19 cells challenged with gp120 as our model system. METHODS: Several analytical techniques were employed to decipher the intricate interactions at play. These included PCR, Western blot, and immunofluorescence assays for the molecular characterization, and transendothelial electrical resistance (TEER) measurements to evaluate barrier integrity. RESULTS: We observed that AEG-1 expression was elevated, whereas the expression levels of tight junction proteins ZO-1, Occludin, and Claudin5 were downregulated in gp120-challenged cells. TEER measurements corroborated these findings, indicating barrier dysfunction. Additional mechanistic studies revealed that the activation of NFκB and MMP2/9 pathways mediated the AEG-1-induced barrier destabilization. Through the use of lentiviral vectors, we engineered cell lines with modulated AEG-1 expression levels. Silencing AEG-1 alleviated gp120-induced downregulation of tight junction proteins and barrier impairment while concurrently inhibiting the NFκB and MMP2/9 pathways. Conversely, overexpression of AEG-1 exacerbated these pathological changes, further compromising the integrity of the BRB. CONCLUSION: Gp120 upregulates the expression of AEG-1 and activates the NFκB and MMP2/9 pathways. This in turn leads to the downregulation of tight junction proteins, resulting in the disruption of barrier function.
Asunto(s)
Barrera Hematorretinal , Proteína gp120 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , Proteínas de la Membrana , Proteínas de Unión al ARN , Humanos , Barrera Hematorretinal/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the neuropathologic progression of HAND. A recent study by our group found that gp120 mediates A1 astrocytes (neurotoxicity), which secrete proinflammatory factors and promote HAND disease progression. Here, by comparing the expression of A2 astrocyte (neuroprotective) markers in the brains of gp120 tgm mice and gp120+/α7nAChR-/- mice, we found that inhibition of alpha 7 nicotinic acetylcholine receptor (α7nAChR) promotes A2 astrocyte generation. Notably, kynurenine acid (KYNA) is an antagonist of α7nAChR, and is able to promote the formation of A2 astrocytes, the secretion of neurotrophic factors, and the enhancement of glutamate uptake through blocking the activation of α7nAChR/NF-κB signaling. In addition, learning, memory and mood disorders were significantly improved in gp120 tgm mice by intraperitoneal injection of kynurenine (KYN) and probenecid (PROB). Meanwhile, the number of A2 astrocytes in the mouse brain was significantly increased and glutamate toxicity was reduced. Taken together, KYNA was able to promote A2 astrocyte production and neurotrophic factor secretion, reduce glutamate toxicity, and ameliorate gp120-induced neuropathological deficits. These findings contribute to our understanding of the role that reactive astrocytes play in the development of HAND pathology and provide new evidence for the treatment of HAND via the tryptophan pathway.
Asunto(s)
Astrocitos , Ácido Glutámico , Quinurenina , Animales , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Ratones , Quinurenina/metabolismo , Ácido Quinurénico/metabolismo , Ácido Quinurénico/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/toxicidad , Transducción de Señal/efectos de los fármacos , Ratones Noqueados , Probenecid/farmacología , Ratones Endogámicos C57BL , Masculino , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/efectos de los fármacos , FN-kappa B/metabolismoRESUMEN
Apart from mediating viral entry, the function of the free HIV-1 envelope protein (gp120) has yet to be elucidated. Our group previously showed that EP2 derived from one ß-strand in gp120 can form amyloid fibrils that increase HIV-1 infectivity. Importantly, gp120 contains ~30 ß-strands. We examined whether gp120 might serve as a precursor protein for the proteolytic release of amyloidogenic fragments that form amyloid fibrils, thereby promoting viral infection. Peptide array scanning, enzyme degradation assays, and viral infection experiments in vitro confirmed that many ß-stranded peptides derived from gp120 can indeed form amyloid fibrils that increase HIV-1 infectivity. These gp120-derived amyloidogenic peptides, or GAPs, which were confirmed to form amyloid fibrils, were termed gp120-derived enhancers of viral infection (GEVIs). GEVIs specifically capture HIV-1 virions and promote their attachment to target cells, thereby increasing HIV-1 infectivity. Different GAPs can cross-interact to form heterogeneous fibrils that retain the ability to increase HIV-1 infectivity. GEVIs even suppressed the antiviral activity of a panel of antiretroviral agents. Notably, endogenous GAPs and GEVIs were found in the lymphatic fluid, lymph nodes, and cerebrospinal fluid (CSF) of AIDS patients in vivo. Overall, gp120-derived amyloid fibrils might play a crucial role in the process of HIV-1 infectivity and thus represent novel targets for anti-HIV therapeutics.
Asunto(s)
Amiloide , Proteína gp120 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Humanos , Amiloide/metabolismo , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Proteínas Amiloidogénicas/metabolismo , Virión/metabolismo , Péptidos/metabolismo , Péptidos/química , Péptidos/farmacologíaRESUMEN
The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332, but Asn at this position is not absolutely conserved or required for HIV-1 entry based on the prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes, with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity with high levels of cell surface expression and slightly higher gp120 shedding than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs were in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states. IMPORTANCE: Glycan attached to amino acid asparagine at position 332 of HIV-1 envelope glycoproteins is a main target of a subset of broadly neutralizing antibodies that block HIV-1 infection. Here, we defined the contribution of different amino acids at this position to Env antigenicity, stability on ice, and conformational states.
Asunto(s)
Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , Aminoácidos , Anticuerpos Neutralizantes , Anticuerpos ampliamente neutralizantes , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Glicoproteínas , Anticuerpos Anti-VIH , Proteína gp120 de Envoltorio del VIH/genética , Hielo , PolisacáridosRESUMEN
HIV envelope protein gp120 is considered a primary molecular determinant of viral neutralization phenotype due to its critical role in viral entry and immune evasion. The intrinsically disordered regions (IDRs) in gp120 are responsible for their extensive sequence variations and significant structural rearrangements. Despite HIV neutralization phenotype and sequence/structural information of gp120 have been experimentally characterized, there remains a gap in our understanding of the correlation between the viral phenotype and IDRs in gp120. Here, we combined machine learning (ML) techniques and molecular dynamics (MD) simulations to gain data-driven and molecule-mechanism insights into relationships between viral sequence, structure, and phenotypes from the perspective of IDRs in gp120. ML models, trained only on the length and disorder score of IDRs, achieved equivalent performance to the best baseline model using amino acid sequences to discriminate HIV neutralization phenotype, indicating that the lengths or disorder of specific IDRs are strongly related to HIV neutralization phenotypes. Comparative MD analysis reveals that gp120 with extreme neutralization phenotypes in multiple conformational states, especially some IDRs, exhibit significantly distinct structural dynamics, conformational flexibility, and thermodynamic distributions. Taken together, our study provided insights into the role of IDRs in gp120 responding to HIV neutralization phenotypes, which will advance the understanding of molecular mechanisms underlying viral function associated with HIV neutralization phenotype and help develop antiviral vaccines or drugs.
Asunto(s)
Proteína gp120 de Envoltorio del VIH , Infecciones por VIH , Humanos , Proteína gp120 de Envoltorio del VIH/genética , Conformación Proteica , Secuencia de Aminoácidos , Fenotipo , Pruebas de NeutralizaciónRESUMEN
Dendritic cells (DCs) play a crucial role in HIV-1 infection of CD4+ T cells. DC-SIGN, a lectin expressed on the surface of DCs, binds to the highly mannosylated viral membrane protein gp120 to capture HIV-1 virions and then transport them to target T cells. In this study, we modified peptide C34, an HIV-1 fusion inhibitor, at different sites using different sizes of the DC-SIGN-specific carbohydrates to provide dual-targeted HIV inhibition. The dual-target binding was confirmed by mechanistic studies. Pentamannose-modified C34 inhibited virus entry into both DC-SIGN+ 293T cells (52%-71% inhibition at 500 µM) and CD4+ TZM-b1 cells (EC50 = 0.7-1.7 nM). One conjugate, NC-M5, showed an extended half-life relative to C34 in rats (T1/2: 7.8 vs 1.02 h). These improvements in antiviral activity and pharmacokinetics have potential for HIV treatment and the development of dual-target inhibitors for pathogens that require the involvement of DC-SIGN for infection.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Animales , Ratas , Línea Celular , VIH-1/metabolismo , Lectinas Tipo C/metabolismo , Células Dendríticas/metabolismo , Polisacáridos/farmacología , Proteína gp120 de Envoltorio del VIH/metabolismoRESUMEN
BACKGROUND & AIMS: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-ß1 signaling. METHODS: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. RESULTS: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-ß1 (TGF-ß1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-ß1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-ß1-induced fibrogenesis. CONCLUSIONS: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-ß1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. IMPACT AND IMPLICATIONS: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-ß1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.
Asunto(s)
Coinfección , Infecciones por VIH , Virus de la Hepatitis B , Subunidad alfa del Factor 1 Inducible por Hipoxia , Cirrosis Hepática , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Cirrosis Hepática/metabolismo , Cirrosis Hepática/virología , Cirrosis Hepática/patología , Humanos , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Virus de la Hepatitis B/genética , Coinfección/virología , Ratones Endogámicos C57BL , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Hepatitis B Crónica/virología , Proteína gp120 de Envoltorio del VIH/metabolismo , Hepatocitos/metabolismo , Hepatocitos/virología , Hepatocitos/patología , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/virología , Modelos Animales de Enfermedad , Células Hep G2 , MasculinoRESUMEN
The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.
Asunto(s)
Anticuerpos ampliamente neutralizantes , Infecciones por VIH , Humanos , Aminoácidos , Anticuerpos ampliamente neutralizantes/inmunología , Antígenos CD4/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Epítopos , Anticuerpos Anti-VIH , Antígenos VIH , Proteína gp120 de Envoltorio del VIH/genética , Seropositividad para VIH , VIH-1/genética , Vacunas contra el SIDA/inmunologíaRESUMEN
Liver fibrosis is the excessive accumulation of extracellular matrix proteins, primarily collagen, in response to liver injury caused by chronic liver diseases. HIV infection accelerates the progression of liver fibrosis in patients co-infected with HCV or HBV compared to those who are only mono-infected. The early event in the progression of liver fibrosis involves the activation of hepatic stellate cells (HSCs), which entails the loss of lipid droplets (LD) to fuel the production of extracellular matrix components crucial for liver tissue healing. Thus, we are examining the mechanism by which HIV stimulates the progression of liver fibrosis. HIV-R5 tropic infection was unable to induce the expression of TGF-ß, collagen deposition, α-smooth muscle actin (α-SMA), and cellular proliferation. However, this infection induced the secretion of the profibrogenic cytokine IL-6 and the loss of LD. This process involved the participation of peroxisome proliferator-activated receptor (PPAR)-α and an increase in lysosomal acid lipase (LAL), along with the involvement of Microtubule-associated protein 1 A/1B-light chain 3 (LC3), strongly suggesting that LD loss could occur through acid lipolysis. These phenomena were mimicked by the gp120 protein from the R5 tropic strain of HIV. Preincubation of HSCs with the CCR5 receptor antagonist, TAK-779, blocked gp120 activity. Additionally, experiments performed with pseudotyped-HIV revealed that HIV replication could also contribute to LD loss. These results demonstrate that the cross-talk between HSCs and HIV involves a series of interactions that help explain some of the mechanisms involved in the exacerbation of liver damage observed in co-infected individuals.