RESUMEN

[This corrects the article DOI: 10.1371/journal.pone.0181886.].

RESUMEN

We describe the properties of BG505 SOSIP.664 HIV-1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native-like trimers that are the basis for many structure-guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV-1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum-free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size-exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10 . The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native-like as judged by negative-stain electron microscopy, and were stable over a multi-month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native-like Env glycoprotein trimers of various designs and genotypes.

Asunto(s)

Vacunas contra el SIDA/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas contra el SIDA/genética , Animales , Anticuerpos Neutralizantes/inmunología , Células CHO , Cricetulus , Glicosilación , Infecciones por VIH/virología , Humanos , Multimerización de Proteína , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genética

RESUMEN

Rabbits and monkeys immunized with HIV type 1 (HIV-1) native-like BG505 SOSIP.664 (BG505s) glycoprotein trimers are known to induce antibodies that can neutralize the autologous tier-2 virus. Here, we assessed the induction of HIV-1 trimer binding and neutralizing antibody (nAb) titres when BG505s trimers were also delivered by non-replicating simian (chimpanzee) adenovirus and non-replicating poxvirus modified vaccinia virus Ankara (MVA) vaccine vectors. First, we showed that approximately two-thirds and one-third of the trimers secreted from the ChAdOx1.BG505s (C) and MVA.BG505s (M) vaccine-infected cells, respectively, were cleaved and in a native-like conformation. Rabbits were immunized intramuscularly with these vaccine vectors and in some cases boosted with ISCOMATRIX™-adjuvanted BG505s protein trimer (P), using CCC, MMM, PPP, CPP, MPP and CMP vaccine regimens. We found that the peak trimer-binding antibody and tier-1A and autologous tier-2 nAb responses induced by the CC, CM, PPP, CPP, MPP and CMP regimens were comparable, although only PPP induced autologous tier-2 nAbs in all the immunized animals. Three animals developed weak heterologous tier-2 nAbs. These results demonstrate that ChAdOx1 and MVA vectors are useful delivery modalities for not only T-cell, but also antibody vaccine development.

Asunto(s)

Adenovirus de los Simios/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Virus Vaccinia/inmunología , Vacunas Virales/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/inmunología , Animales , Línea Celular , Células HEK293 , Humanos , Multimerización de Proteína/inmunología , Conejos , Vacunación , Vacunas de ADN

RESUMEN

The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Polisacáridos/metabolismo , Anticuerpos Neutralizantes/inmunología , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Glicopéptidos/análisis , Glicosilación , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Polisacáridos/análisis , Polisacáridos/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

HIV-1 variants resistant to small molecule CCR5 inhibitors recognize the inhibitor-CCR5 complex, while also interacting with free CCR5. The most common genetic route to resistance involves sequence changes in the gp120 V3 region, a pathway followed when the primary isolate CC1/85 was cultured with the AD101 inhibitor in vitro, creating the CC101.19 resistant variant. However, the D1/86.16 escape mutant contains no V3 changes but has three substitutions in the gp41 fusion peptide. By using CCR5 point-mutants and gp120-targeting agents, we have investigated how infectious clonal viruses derived from the parental and both resistant isolates interact with CCR5. We conclude that the V3 sequence changes in CC101.19 cl.7 create a virus with an increased dependency on interactions with the CCR5 N-terminus. Elements of the CCR5 binding site associated with the V3 region and the CD4-induced (CD4i) epitope cluster in the gp120 bridging sheet are more exposed on the native Env complex of CC101.19 cl.7, which is sensitive to neutralization via these epitopes. However, D1/86.16 cl.23 does not have an increased dependency on the CCR5 N-terminus, and its CCR5 binding site has not become more exposed. How this virus interacts with the inhibitor-CCR5 complex remains to be understood.

Asunto(s)

Antagonistas de los Receptores CCR5 , Farmacorresistencia Viral/fisiología , VIH-1/fisiología , Interacciones Huésped-Patógeno/fisiología , Leucocitos Mononucleares/virología , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Línea Celular , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/química , VIH-1/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Conejos , Receptores CCR5/inmunología , Receptores CCR5/metabolismo , Alineación de Secuencia , Replicación Viral/efectos de los fármacos , Replicación Viral/genética

RESUMEN

The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.

Asunto(s)

Sustitución de Aminoácidos , Proteínas gp160 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Fusión Celular , Línea Celular , Dimerización , Regulación Viral de la Expresión Génica , VIH-1/genética , VIH-1/metabolismo , VIH-1/patogenicidad , Células HeLa , Humanos , Virión/metabolismo , Virión/patogenicidad

RESUMEN

The HIV-1 envelope glycoprotein is expressed on the viral membrane as a trimeric complex, formed by three gp120 surface glycoproteins non-covalently associated with three membrane-anchored gp41 subunits. The labile nature of the association between gp120 and gp41 hinders the expression of soluble, fully cleaved, trimeric gp140 proteins for structural and immunization studies. Disruption of the primary cleavage site within gp160 allows the production of stable gp140 trimers, but cleavage-defective trimers are antigenically dissimilar from their cleaved counterparts. Soluble, stabilized, proteolytically cleaved, trimeric gp140 proteins can be generated by engineering an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP). We have found that SOSIP gp140 proteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared to a prototypic strain (JR-FL, subtype B). We now show that the determinants of this enhanced stability are located in the N-terminal region of KNH11144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability. The stabilized trimers retain the epitopes for several neutralizing antibodies (b12, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, suggesting that the overall antigenic structure of the gp140 protein has not been adversely impaired by the trimer-stabilizing substitutions. The ability to increase the stability of gp140 trimers might be useful for neutralizing antibody-based vaccine strategies based on the use of this type of immunogen.

Asunto(s)

Productos del Gen env/metabolismo , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Estructura Terciaria de Proteína/fisiología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Inmunoadhesinas CD4/inmunología , Línea Celular , Epítopos/inmunología , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Alineación de Secuencia , Solubilidad , Relación Estructura-Actividad , Productos del Gen env del Virus de la Inmunodeficiencia Humana

RESUMEN

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4(+) T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4(+) T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4(+) T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/inmunología , Cambio de Clase de Inmunoglobulina/genética , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Recombinación Genética/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor Activador de Células B , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD4/metabolismo , Antígenos CD40/metabolismo , Células Cultivadas , Citocinas/farmacología , Humanos , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos , Manosa/metabolismo , Proteínas de la Membrana/inmunología , Unión Proteica , Receptores de Antígenos de Linfocitos B/inmunología , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Bazo/inmunología , Bazo/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba