RESUMEN
The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Fragmentos de Péptidos/inmunología , Multimerización de Proteína/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , VIH-1/clasificación , VIH-1/genética , Humanos , Inmunización , Modelos Moleculares , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Virión/química , Virión/inmunología , Virión/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human immunodeficiency virus type 1 has several genetic subtypes and two coreceptor use phenotypes: R5 that uses CCR5, while X4 uses CXCR4. A high amino acid charge of the envelope glycoprotein 120 V3 region, common at positions 11 and 25, is important for CXCR4 use. We characterized charged V3 amino acids, retrieving all biologically phenotyped sequences from the HIV Sequence Database. Selecting individually unique ones randomly yielded 48 subtype A, 231 B, 180 C, 37 D and 32 CRF01_AE sequences; 482 were R5 and 46 were X4. Charged amino acids were conserved in both R5 and X4 with general and subtype-specific patterns. X4 viruses gained a higher charge from positive amino acids at positions other than in R5, and through the loss of negative amino acids. Other positions than 11/25 had a greater impact on charge (P<0.001). This describes how R5 evolves into X4 in a subtype-specific context, useful for computer-based predictions and vaccine design.
Asunto(s)
Aminoácidos Básicos/química , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Receptores CXCR4/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos Básicos/genética , Aminoácidos Básicos/metabolismo , Línea Celular , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/química , VIH-1/clasificación , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores del VIH/genética , Receptores del VIH/metabolismo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Plasma cells (PCs) are defined as terminally differentiated B cells that secrete large amounts of immunoglobulin (Ig). PCs that reside in the bone marrow (BM) are responsible for maintaining long-term antibody (Ab) responses after infection and vaccination, while PCs present in the blood are generally short-lived. In rhesus macaques, a species frequently used for the evaluation of human vaccines, B cells resemble those found in humans. However, a detailed characterization of BM-resident rhesus PC phenotype and function is lacking. Here, we examined Ig secretion of distinct rhesus CD138+ populations by B cell ELISpot analysis to couple phenotype with function. We demonstrate that the CD20low/-CD138+CD31+ BM population was highly enriched for antibody-secreting cells with IgG being the predominant isotype (60%), followed by IgA (33%) and IgM (7%). Transmission electron microscopy analysis confirmed PC enrichment in the CD20low/-CD138+CD31+ population with cells containing nuclei with "spokes of a wheel" chromatin structure and prominent rough endoplasmic reticulum. This panel also stained human BM PCs and allowed a clear distinction between BM PCs and short-lived peripheral PCs, providing an improved strategy to isolate PCs from rhesus BM for further analysis.
RESUMEN
Human immunodeficiency virus type 1 (HIV-1) enters cells through the chemokine receptors CCR5 (R5 virus) and/or CXCR4 (X4 virus). Loss of N-linked glycans and increased net charge of the third variable loop (V3) of the gp120 envelope glycoprotein have been observed to be important steps towards CXCR4 use. All reported sequences using CCR5 or CXCR4 exclusively, or using both, were gathered from the Los Alamos HIV Database and analysed with regard to the V3 N-linked glycosylation motifs (sequons) and charge. The V3 loop glycan had a sensitivity of 0.98 and a 0.92 positive predictive value in the context of CCR5 use. The difference from X4 was remarkable (P<10(-12)). Especially, the sequon motif NNT within the V3 loop was conserved in 99.2 % of the major clades. The results suggest a close association between the V3 loop glycan and CCR5 use and may provide new insight into HIV-1 tropism and help to improve phenotype-prediction models.