the conserved CD4 binding site (CD4bs) are among the most broad and potent. The VRC01 class of CD4bs antibodies derive from a VH1-2 germline gene and have a characteristic short (5AA) CDRL3. These antibodies are highly affinity matured and when reverted to germline sequence fail to recognize most HIV Env sequences. Thus, a major challenge is to design immunogens that would activate the appropriate naïve B cells and generate VRC01 class antibodies. Previous studies have identified few clade C gp140 and clade B gp120 outer domain constructs (OD) that can engage germline of a subset of CD4bs antibodies. Here, we aim to identify HIV clade A OD mutants that can bind to germlines of diverse CD4bs antibodies to be used as HIV imunogens. Methods: Clade A OD4.2.2 (PDB code 4I3R) was displayed on the cell surface using a yeast display system. Mutations in its D loop (274-283), which constitutes part of the CD4bs, were introduced by PCR to construct two libraries: one with randomized sequences and the other with sequences designed by structure based bioinformatics. The libraries were screened for binding to germline revertants of VRC01-class antibodies. Germline binders were sorted with FACS and characterized. Results: Consistent with previous studies, the original yeast displayed OD failed to bind to any of the germline revertants of CD4bs antibodies. Two clones from the randomized D loop library were identified to bind VRC03 germline (VH1-2). When displayed on lumazine synthase 60mer nanoparticles, they bound to several VRC01 class germline revertants including VRC01, VRC07, VRC20, 12A12, CH31 and VRC03. Conclusions: Optimization of amino acids in loop D of the OD helped to identify mutants that can bind to diverse VRC01 class germline reverted antibodies. These OD constructs can thus serve as priming immunogens to elicit CD4bs bNab.Background: The recently published structures of the clade Aderived BG505 SOSIP trimers, mimetics of the native HIV envelope glycoprotein (Env) spike, mark the beginning of new era in HIV structural biology. Displaying a well-ordered quaternary array, the BG505 SOSIP trimers display an excellent antigenic profile, discriminating recognition by broadly neutralizing antibodies (bNAbs) from non-broadly neutralizing antibodies (non-bNAbs) and represent interesting Env-based immunogens. Even with this significant advance, obtaining soluble SOSIP trimers derived from other clades has been challenging. Here, we report the isolation of two SOSIP trimers derived from clades B and C. Methods: We modified the 2G12 affinity purification by replacement with lectin affinity purification, affording a scalable process with mild elution conditions. Using CD4 binding sitedirected (CD4bs) non-bNAbs in a negative selection purification process, we obtained homogeneous well-ordered clade B JRFL
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