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
A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Sitios de Unión de Anticuerpos/genética , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/ultraestructura , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Evasión Inmune/genética , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes/aislamiento & purificación , Sitios de Unión de Anticuerpos/inmunología , Antígenos CD4/farmacología , Línea Celular Tumoral , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Epítopos/inmunología , Glicosilación , Anticuerpos Anti-VIH/aislamiento & purificación , Antígenos VIH/genética , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/inmunología , VIH-1/genética , Células HeLa , Humanos , Evasión Inmune/inmunología , Pruebas de Neutralización , Proteínas Recombinantes/farmacologíaRESUMEN
UNLABELLED: Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. IMPORTANCE: The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.
Asunto(s)
VIH-1/inmunología , VIH-1/fisiología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Sustitución de Aminoácidos , Anticuerpos Anti-VIH , Antígenos VIH/química , Antígenos VIH/genética , Antígenos VIH/ultraestructura , VIH-1/genética , Humanos , Microscopía Electrónica de Transmisión , Modelos Moleculares , Imitación Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The matrix protein p17gag (MA) is a product of proteolytic cleavage of the gag gene encoded polyprotein (pr55gag) and is formed when HIV particles undergo the process of maturation. The MA protein is associated with the inner surface of the viral membrane and determines the overall shape of the virion. Previous studies have shown the existence of trimers of MA in solution and in the crystalline state. Here, we used molecular modelling methods to identify feasible interactions between pairs of MA trimers and have related this to structural data from electron microscopy. A systematic search docking procedure was able to identify many energetically favourable conformations for a pair of trimers, including some which have been previously reported. These conformations were used to generate several networks of MA trimers, which were then evaluated against structural observations of the MA network. The model suggested here provides a good match with experimental data such as the spacing between gag protein rings, the number and disposition of glycoprotein (gp41-gp120) knobs and the number of copies of MA in a virus particle. It also rationalizes the observed distribution of sizes of virus particles and is consistent with the presence of icosahedral organisation in mature HIV. Energy minimisation performed with explicit water and counter ions, was used to identify residues participating in inter-trimer interactions. The nature of these interactions is discussed in relation to the conservation of these residues in reported variants of the HIV and SIV MA protein sequences.