Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies

Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti–CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions

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

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs

Amino acid alignment of b12 mature and germline heavy and light chain variable regions.

<p>The framework (FR) and complementary determining regions (CDR) are outlined, and the D- and J- gene segments are boxed. The complementarity determining (CDR) and framework (FW) regions were determined using the IMGT/V-Quest tool (<a href="http://www.imgt.org" target="_blank">www.imgt.org</a>). Amino acid numbering is based on the Kabat numbering system. Seven amino acids (two in the VD joining region and five in the DJ joining region) that are present in the mature sequence (and which were left unchanged in the germline sequence used here) are shown in oval. The four amino acids in the VH chain that are known to make direct contact with the gp120 core are highlighted in yellow.</p

Effect of germline-to mature VH mutations on the binding and neutralizing activity of IgG b12 expressing the mature VL chain.

<p>(<b>A</b>) The S31, A52P and G53Y, G100W, and S31N/A52P/G53Y/G100W amino acid mutations were introduced on the germline VH chain. The mutated heavy chains were combined with the mature light chains and the corresponding IgGs were made. Their binding to the indicated recombinant Envs was determined. The binding of the fully mature b12, that of the germline b12 and that of the gHC/mLC chimera were also determined. The shaded grey area indicates background (non-specific) binding (determined as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003106#s4" target="_blank">Materials and Methods</a> section). All binding curves are representative from two to four independent experiments. (<b>B</b>) Neutralizing activities of the same IgGs against the indicated virions.</p

Kinetic parameters of the mature and chimeric IgG b12 forms for the QH0692 gp120.

<p>Kinetic parameters of the mature and chimeric IgG b12 forms for the QH0692 gp120.</p

Binding of mature, germline and chimeric IgG b12 forms to the indicated recombinant Env was determined by ELISA as described in the

<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003106#s4" target="_blank"><b>Materials and Methods</b></a><b> section.</b> (−): no binding; (+): binding; gp140(t): trimeric gp140; gp140(m): monomeric gp140.</p

Env-specific B-cell activation through mature and chimeric b12 BCRs.

<p>(<b>A</b>) Cell-surface expression of b12 mature, germline and chimeric BCRs on the surface of B cells. (<b>B</b>) Intracellular Ca²⁺ flux mediated by the mature, germline and chimeric BCRs following BCRs cross-linking by goat anti-human IgG (H+L) F(ab′)₂ in A20 cells. (<b>C</b>) Binding of mature, germline and chimeras to SF162 gp140 trimer. (<b>D</b>) Intracellular Ca²⁺ flux upon addition of SF162 gp140 trimers to B cells (DG-75) expressing the indicated b12 BCRs. (<b>E</b>) Binding of mature, germline and chimeras to QH0692 gp140 trimer. (<b>F</b>) Intracellular Ca²⁺ flux upon addition of QH0692 gp140 trimers to B cells expressing the indicated b12 BCRs.</p

Binding and neutralizing properties of the b12 mature, germline and mature/germline b12 chimeras to Env.

<p>(<b>A</b>) Kinetic analysis of binding of mature b12 and of the two chimeras to QH0692 gp120. The Env concentrations tested are shown. A summary of the results is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003106#ppat-1003106-t001" target="_blank"><b>Table 1</b></a>. (<b>B</b>) IgG-binding to QH0692, SF162 and HXB2 gp120s by ELISA. The shaded grey area indicates background (non-specific) binding (determined as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003106#s4" target="_blank">Materials and Methods</a> section). All binding curves are representative from two to four independent experiments. (<b>C</b>) Neutralizing activities of IgG against QH0692, SF162 and HXB2 virions. Results are representative from two to four independent experiments.</p