N332-Directed Broadly Neutralizing Antibodies Use Diverse Modes of HIV-1 Recognition: Inferences from Heavy-Light Chain Complementation of Function

<div><p>Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332) is characterized by an <i>N</i>-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type) was generally consistent (58±10 µg/ml). In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.</p> </div

Heavy and light chain nucleotide sequence diversity derived from phylogenetic analysis of PGT121-137 antibodies.

<p>Neighbor-joining (NJ) method was used to calculate the dendrogram. Antibody branches are color-coded according to donors with putative V and J genes labeled under the donor specification.</p

DFIRE scores of homology modeling of PGT121-137, based on PGT128, PGT121 and 2G12 template structures.

<p>Sequences alignment and homology modeling were carried out with sequences for PGT121-137 heavy and light chain and PGT128, PGT121 and 2G12 structural templates. Low scores indicate good homology modeling and were lowest for the PGT128 sequence modeled on the PGT128 structure (or PGT121 sequence modeled on PGT121 structure). DFIRE score <0.4 are shown in red, 0.4–0.45 in yellow and 0.45–0.5 in green.</p

Relative interdonor complementation for N332-directed antibodies versus VRC01-like antibodies.

<p>Relative neutralization of inter and intra-donor chimeric antibodies. The neutralization of the chimeric antibodies relative to the average neutralization of the wild-type antibodies for the heavy and light chain components is plotted for the N332-directed antibodies (produced by 96-well microplate-formatted expression method) and VRC01-like antibodies (produced by small scale protein production method). The mean N332 intra-donor relative neutralization is 0.82±0.4 while the inter-donor mean is far lower at 0.11±0.16 revealing a functional complementation in the intra-donor chimeras that is not seen with the inter-donor antibodies (p<0.0001). Conversely the VRC01-like antibodies display similar results for intra (0.61±0.45) and inter-donor (0.66±0.43) complementation (p = 0.58). Neutralization values of VRC01-like chimeras were taken from 50 µg/ml concentration points used in previously published neutralization titers (31). Results for strains in which the wild-type neutralization was below 50% were excluded from the analysis.</p

ELISA binding to HIV-1 gp120 by PGT121-137 antibodies and chimeric variants in units of OD₄₅₀

<p> <b>nm.</b> Strong binding is coded in red (>1.0 OD450 nm), intermediate in yellow (0.5–1.0 OD450 nm) and weak in green (0.3–0.5 OD450 nm). Each reported value is the average from three individual measurements, with the absorbance normalized by the expression titer. Note that in cases when the expression level of the antibodies was more than 2-fold different from the average, the normalization is probably not accurate and these numbers have been marked with a star. For comparison, the raw ELISA data has been added as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055701#pone.0055701.s004" target="_blank">Figure S4</a>.</p

Neutralization by PGT121-137 antibodies and chimeric variants.

<p>Strong neutralization is coded in red (>90%), intermediate in yellow (75–90%) and weak in green (50–75%). Neutralization data were generated in single neutralization experiments, with each test sample analyzed in duplicate to determine the percent decrease in virus growth (neutralization) as compared and normalized to the control wells without antibody expression (defined as 0% neutralization). Each antibody was assayed at a 1∶5 final dilution with virus added. Neutralization results were normalized by the relative antibody expression, with the maximum neutralization defined to be 100%. Note that in cases when the expression level of the antibodies was more than 2-fold different from the average, the normalization is probably not accurate and these numbers have been marked with a star. For comparison, the raw neutralization data has been added as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055701#pone.0055701.s005" target="_blank">Figure S5</a>. Supernatants expressing antibody 17b showed 0% neutralization with HIV-1 TRO.11 and 27% neutralization with HIV-1 SF162.LS; supernatants expressing VRC01 showed 100% neutralization to both HIV-1 viruses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055701#pone.0055701.s008" target="_blank">Figure S8</a>).</p

Transplanting Supersites of HIV-1 Vulnerability

<div><p>One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of “supersite transplants”, capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose₉-<i>N</i>-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 <i>N</i>-linked glycans and ∼25 Env residues, can be segregated into acceptor scaffolds away from the immune-evading capabilities of the rest of HIV-1 Env, thereby providing a means to focus the immune response on the scaffolded supersite.</p></div