Engineering, expression, purification, and characterization of stable clade A/B recombinant soluble heterotrimeric gp140 proteins.
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.
Seattle BioMed, Seattle, Washington, USA.
George
G
Sellhorn
Zane
Z
Kraft
Zachary
Z
Caldwell
Katharine
K
Ellingson
Christine
C
Mineart
Michael S
MS
Seaman
David C
DC
Montefiori
Eliza
E
Lagerquist
Leonidas
L
Stamatatos
D000818:Animals; D002460:Cell Line; D019281:Dimerization; D015870:Gene Expression; D015483:HIV Antibodies / Q000276:immunology; D015658:HIV Infections / Q000276:immunology / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D015202:Protein Engineering; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
D015483:HIV Antibodies; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1