Engineering, Expression, Purification, and Characterization of Stable Clade A/B Recombinant Soluble Heterotrimeric gp140 Proteins

Abstract

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is composed of two noncovalently associated subunits: an extracellular subunit (gp120) and a transmembrane subunit (gp41). The functional unit of Env on the surface of infectious virions is a trimer of gp120/gp41 heterodimers. Env is the target of anti-HIV neutralizing antibodies. A considerable effort has been invested in the engineering of recombinant soluble forms of the virion-associated Env trimer as vaccine candidates to elicit anti-HIV neutralizing antibody responses. These soluble constructs contain three gp120 subunits and the extracellular segments of the corresponding gp41 subunits. The individual gp120/gp41 protomers on these soluble trimers are identical in amino acid sequence (homotrimers). Here, we engineered novel soluble trimeric gp140 proteins that are formed by the association of gp140 protomers that differ in amino acid sequence and glycosylation patterns (heterotrimers). Specifically, we engineered soluble heterotrimeric proteins composed of clade A and clade B Env protomers. The clade A gp140 protomers were derived from viruses isolated during acute infection (Q168a2, Q259d2.17, and Q461e2), whereas the clade B gp140 protomers were derived from a virus isolated during chronic infection (SF162). The amino acid sequence divergence between the clade A and the clade B Envs is approximately 24%. Neutralization epitopes in the CD4 binding sites and coreceptor binding sites, as well as the membrane-proximal external region (MPER), were differentially expressed on the heterotrimeric and homotrimeric proteins. The heterotrimeric gp140s elicited broader anti-tier 1 isolate neutralizing antibody responses than did the homotrimeric gp140s.

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate

“…Although the neutralization activity was limited to Tier 1 pseudoviruses, this result is in agreement with other immunogenicity studies performed with soluble HIV Envelope proteins of different clades with our data even suggesting improved neutralization in several instances [39-42]. Moreover, it is prospected to re-evaluate the breadth of neutralization activity in A3R5 cells, which have been recently shown to be more sensitive to neutralization than the TZM-bl used in the present study [43].…”

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

“…IgGs were produced by transient transfection of HEK 293e cells suspension cells, as previously described [53], [54] with a few modifications. Briefly, cells were suspended in FreeStyle 293 expression medium (Invitrogen, Carlsbad, CA) to a density of 20 million cells/mL.…”

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

“…With the increasing understanding of the architecture of the viral spike (21,76,77), the possibility to generate stable soluble trimers that closely resemble the native spike (78,79), and the means to generate structurally arrested peptide mimetics of gp120 microdomains (31), a number of tools have become available which have promise to tailor future DARPin selections to specific domains of interest. As discussed above, based on our current data, this holds particular promise to improve envelope-specific DARPin identification and to harness the distinctive binding properties of DARPins for HIV inhibitor development.…”