RESUMEN
Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.
Asunto(s)
VIH-1/química , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/inmunología , Reactivos de Enlaces Cruzados , Microscopía por Crioelectrón , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/química , Antígenos VIH/inmunología , Antígenos VIH/ultraestructura , Interacciones Huésped-Patógeno/inmunología , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Conejos , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructuraRESUMEN
The clinical relevance of the transient intraluminal filament model of middle cerebral artery occlusion (tMCAO) has been questioned due to distinct cerebral blood flow profiles upon reperfusion between tMCAO (abrupt reperfusion) and alteplase treatment (gradual reperfusion), resulting in differing pathophysiologies. Positive results from recent endovascular thrombectomy trials, where the occluding clot is mechanically removed, could revolutionize stroke treatment. The rapid cerebral blood flow restoration in both tMCAO and endovascular thrombectomy provides clinical relevance for this pre-clinical model. Any future clinical trials of neuroprotective agents as adjuncts to endovascular thrombectomy should consider tMCAO as the model of choice to determine pre-clinical efficacy.
