RESUMEN
A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Proteína gp41 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , Macaca mulatta , Animales , Humanos , Proteína gp41 de Envoltorio del VIH/inmunología , Anticuerpos Anti-VIH/inmunología , Ratones , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Vacunación , Anticuerpos ampliamente neutralizantes/inmunología , Linfocitos B/inmunología , Nanopartículas/química , Femenino , Regiones Determinantes de Complementariedad/inmunología , Epítopos/inmunologíaRESUMEN
Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Anticuerpos Bloqueadores/química , Anticuerpos Bloqueadores/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Conformación Proteica , Resonancia por Plasmón de Superficie , Difracción de Rayos XRESUMEN
Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.
Asunto(s)
Diseño de Fármacos , Epítopos/química , Epítopos/inmunología , Estabilidad Proteica , Vacunas contra Virus Sincitial Respiratorio/química , Vacunas contra Virus Sincitial Respiratorio/inmunología , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Macaca mulatta/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Pruebas de Neutralización , Conformación Proteica , Virus Sincitiales Respiratorios/química , Virus Sincitiales Respiratorios/inmunologíaRESUMEN
We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.
Asunto(s)
Células de la Médula Ósea/patología , Antígeno CD11b/metabolismo , Capilares/patología , Diabetes Mellitus Experimental/patología , Leucostasis/patología , Monocitos/patología , Receptores CCR5/sangre , Traslado Adoptivo , Animales , Receptor 1 de Quimiocinas CX3C , Capilares/efectos de los fármacos , Capilares/metabolismo , Capilares/fisiopatología , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Recuento de Células , Movimiento Celular/efectos de los fármacos , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/fisiopatología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Fluorescencia , Fluorobencenos/farmacología , Leucostasis/sangre , Leucostasis/complicaciones , Leucostasis/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pirimidinas/farmacología , Receptores CCR2/metabolismo , Receptores de Quimiocina/metabolismo , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología , Rosuvastatina Cálcica , Estreptozocina , Sulfonamidas/farmacologíaRESUMEN
Diabetic retinopathy (DR) is a leading cause of vision loss among working-age adults. Retinal endothelial cell apoptosis is an early event in DR, and oxidative stress is known to play an important role in this pathology. Recently, we found that high glucose induces apoptosis in retinal neural cells by a caspase-independent mechanism. Here, we investigated the mechanisms underlying retinal endothelial cell apoptosis induced by high glucose and oxidative/nitrosative stress conditions. Endothelial cells (TR-iBRB2 rat retinal endothelial cell line) were exposed to high glucose (long-term exposure, 7 days), or to NOC-18 (nitric oxide donor; 250microM) or H(2)O(2) (100microM) for 24h. Cell viability was assessed by the MTT assay and cell proliferation by [methyl-(3)H]-thymidine incorporation into DNA. Apoptotic cells were detected with Hoechst or Annexin V staining. Active caspases were detected by an apoptosis detection kit. Active caspase-3 and apoptosis-inducing factor (AIF) protein levels were assessed by Western blot or immunohistochemistry. High glucose, NOC-18 and H(2)O(2) increased apoptosis in retinal endothelial cells. High glucose and mannitol decreased cell proliferation, but mannitol did not induce apoptosis. Caspase activation did not increase in high glucose- or NOC-18-treated cells, but it increased in cells exposed to H(2)O(2). However, the protein levels of AIF decreased in mitochondrial fractions and increased in nuclear fractions, in all conditions. These results are the first demonstrating that retinal endothelial cell apoptosis induced by high glucose is independent of caspase activation, and is correlated with AIF translocation to the nucleus. NOC-18 and H(2)O(2) also activate a caspase-independent apoptotic pathway, although H(2)O(2) can also induce caspase-mediated apoptosis.