Article

Construction and characterization of soluble, cleaved, and stabilized trimeric Env proteins based on HIV type 1 Env subtype A.

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
AIDS Research and Human Retroviruses (Impact Factor: 2.71). 07/2006; 22(6):569-79. DOI: 10.1089/aid.2006.22.569
Source: PubMed

ABSTRACT The generation of an antibody response capable of neutralizing a broad range of clinical isolates remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Envelope glycoprotein (Env)-based vaccine candidates will also need to take into account the extensive genetic diversity of circulating HIV-1 strains. We describe here the generation of soluble, stabilized, proteolytically cleaved, trimeric forms of Env (SOSIP gp140 proteins) based on contemporary Env subtype A viruses from East Africa. We discuss issues associated with the construction, purification, and characterization of such complex proteins; not all env sequences allow the expression of trimeric proteins. However, stabilized trimers from one such protein, KNH1144 SOSIP gp140, were successfully made. These proteins are now being prepared for preclinical immunogenicity studies.

0 Bookmarks
 · 
72 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Many efforts have been made in the worldwide quest for a prophylactic HIV vaccine to end the AIDS pandemic, but none has yet succeeded. The lessons learned have repeatedly informed us that the traditional or conventional approaches directly using the pathogens or subunits will not be sufficient for an effective HIV/AIDS vaccine. Recent advances in structure-based technology have shown some promise in the quest for a better immunogen in HIV vaccine development. According to the basic binding structural relationship of an antigen and an antibody, structure-based antigen design could bring some hope for the development of an effective vaccine against HIV.
    Current HIV research 09/2013; · 1.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2 or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deleting most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncating most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding-site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.
    Journal of Virology 07/2013; · 5.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The functional HIV-1 envelope glycoprotein (Env) trimer, the target of anti-HIV-1 neutralizing antibodies (Abs), is innately labile and coexists with non-native forms of Env. This lability and heterogeneity in Env has been associated with its tendency to elicit non-neutralizing Abs. Here, we use directed evolution to overcome instability and heterogeneity of a primary Env spike. HIV-1 virions were subjected to iterative cycles of destabilization followed by replication to select for Envs with enhanced stability. Two separate pools of stable Env variants with distinct sequence changes were selected using this method. Clones isolated from these viral pools could withstand heat, denaturants and other destabilizing conditions. Seven mutations in Env were associated with increased trimer stability, primarily in the heptad repeat regions of gp41, but also in V1 of gp120. Combining the seven mutations generated a variant Env with superior homogeneity and stability. This variant spike moreover showed resistance to proteolysis and to dissociation by detergent. Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9. The latter result may reflect a change in glycans on the stabilized Envs. The stabilizing mutations also increased the proportion of secreted gp140 existing in a trimeric conformation. Finally, several Env-stabilizing substitutions could stabilize Env spikes from HIV-1 clades A, B and C. Spike stabilizing mutations may be useful in the development of Env immunogens that stably retain native, trimeric structure.
    PLoS Pathogens 02/2013; 9(2):e1003184. · 8.14 Impact Factor