Soluble hiv-1 envelope glycoprotein trimers

JRFL SOSIP Trimers

[0220]Applicants are characterizating the second generation stable and soluble Env spikes that are faithful mimetics of the native spike than are the first generation foldon trimers. Applicants apply several means of analysis of second generation soluble Env trimers including antigenic profiling, biophysical assessments, EM and crystallography. Applicants are producing modified JRFL SOSIP trimers that are purified by negative selection to generate highly homogenous trimers by EM (FIG. 1) and binding kinetics. The present invention also encompasses negative selection with F015, and non-neutralizing CD4 binding site mabs.

[0221]Currently, Applicants have produced the JRFL SOSIP trimers by transient transfection that contain the previously described gp120-to-gp41 cysteine pair linkage and the gp41 I / P change, which enhances trimer formation. The JRFL SOSIPs also contain a 168 E / K mutation to restore PG9 / 16 binding and are deleted of the MPER to enhance expression, have...

gp140 Native, Flexible Linker (NFL) Trimers

[0230]Due to the structural limitations of the first generation gp140 foldon trimers and their inability so far to elicit broadly neutralizing antibodies, Applicants designed native-like gp140 trimers in which gp120 is covalently attached to gp41 via a flexible linker (native flexible linker trimers termed gp140-NFL). Applicants developed this new trimer design pathway to make soluble mimetics of the native HIV-1 envelope glycoprotein spike for structural, biophysical and antigenic analysis. A subset of these new trimers that are well-ordered and present trimer-specific neutralizing determinants as HIV vaccine candidates to elicit neutralizing antibodies. Applicants expanded these initial designs to clade C and A virus-derived envelope glycoproteins, and to assess these trimers as soluble immunogens. Applicants also assess immunogenicity of these trimers by particulate, high-density array on liposomes or other particles already under develo...

BG505 gp140-NFL2P Trimers

[0285]The data in FIGS. 27-30 suggests that BG505 gp140-NFL2P trimers are well behaved native like and homogeneous trimers and they do not need F105 negative selection.

[0286]FIG. 24 depicts an antigenicity study of BG505 gp140-NFL2P trimers by ELISA: The ELISA plate was pre-coated with anti-His mAbs. The BG505 gp140-NFL2P trimers (taken directly from the SEC trimer fraction and before F105 negative selection) were captured on the plate by the anti-His Abs. The binding of selected panel of broadly neutralizing (bNAbs; b12, 2G12, VRCO1 and PGVO4), quaternary structure dependent or trimer specific bNAbs (VRCO6, PGT145, PGT151 and PG16) and non-neutralizing monoclonal antibodies (F105, b6 and F240) and V3 mAb, 447-D were studied by ELISA. As is evident from the binding curves, BG505-gp140 NFL2P shows very strong binding with all the bNAbs (except for b12 probably because b12 doesn't neutralize BG505) and also with the trimer specific bNAbs but almost no binding ...