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. 2013 Jan;9(1):e1003106.
doi: 10.1371/journal.ppat.1003106. Epub 2013 Jan 3.

Recombinant HIV envelope proteins fail to engage germline versions of anti-CD4bs bNAbs

Sam Hoot  1 , Andrew T McGuire, Kristen W Cohen, Roland K Strong, Lars Hangartner, Florian Klein, Ron Diskin, Johannes F Scheid, D Noah Sather, Dennis R Burton, Leonidas Stamatatos
Affiliations

Recombinant HIV envelope proteins fail to engage germline versions of anti-CD4bs bNAbs

Sam Hoot et al. PLoS Pathog. 2013 Jan.

Erratum in

  • PLoS Pathog. 2013 Jul;9(9). doi:10.1371/annotation/ed7c0148-97eb-4416-824d-6e6d1aaeceef

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Amino acid alignment of b12 mature and germline heavy and light chain variable regions.
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 (www.imgt.org). 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.
Figure 2
Figure 2. Binding of mature, germline and chimeric IgG b12 forms to the indicated recombinant Env was determined by ELISA as described in the
Materials and Methods section. (−): no binding; (+): binding; gp140(t): trimeric gp140; gp140(m): monomeric gp140.
Figure 3
Figure 3. Binding of mature, germline and mature/germline b12 chimeras to Env.
Mature (A), germline (B), chimera 1 (C), or chimera 2 (D), IgGs were immobilized to anti-human IgG FC capture BLI biosensors and Env proteins were in solution at 1.2 µM. The QH0692 and D368R Envs were in gp120 form. The remaining Envs were in gp140 form (either as trimers or as monomers).
Figure 4
Figure 4. Binding and neutralizing properties of the b12 mature, germline and mature/germline b12 chimeras to Env.
(A) 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 Table 1 . (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 Materials and Methods section). All binding curves are representative from two to four independent experiments. (C) Neutralizing activities of IgG against QH0692, SF162 and HXB2 virions. Results are representative from two to four independent experiments.
Figure 5
Figure 5. Binding of mature, germline and chimeric IgG NIH45-46 and 3BNC60 to Env.
Antibody binding to each Env was determined with BLI using the antigen capture method as described in the Materials and Methods section. Symbols as in Figure 2.
Figure 6
Figure 6. Env-specific B-cell activation through mature and chimeric b12 BCRs.
(A) Cell-surface expression of b12 mature, germline and chimeric BCRs on the surface of B cells. (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. (C) Binding of mature, germline and chimeras to SF162 gp140 trimer. (D) Intracellular Ca2+ flux upon addition of SF162 gp140 trimers to B cells (DG-75) expressing the indicated b12 BCRs. (E) Binding of mature, germline and chimeras to QH0692 gp140 trimer. (F) Intracellular Ca2+ flux upon addition of QH0692 gp140 trimers to B cells expressing the indicated b12 BCRs.
Figure 7
Figure 7. Effect of germline-to mature VH mutations on the binding and neutralizing activity of IgG b12 expressing the mature VL chain.
(A) 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 Materials and Methods section). All binding curves are representative from two to four independent experiments. (B) Neutralizing activities of the same IgGs against the indicated virions.
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