Characteristic of ART-naive HIV-1 infected patients.

<p>Characteristic of ART-naive HIV-1 infected patients.</p

Specificity of gp120/CD4 blocking antibodies.

<p>A) The IgG response against Env was evaluated by flow cytometry using MOLT-BaL and uninfected-MOLT cell line. The levels of anti-Env IgG antibodies were calculated as MFI ratio (MOLT-BaL/MOLT). B) Titer of antibodies against the soluble trimer BG505 SOSIP.664 gp140 was determined by ELISA. A correlation analysis between the anti-Env levels and gp120/CD4 blocking activity is shown (Spearman´s correlation). C) A set of anti-Env antibodies, whose reactivity is well-known, was used to evaluate the specificity of the antibodies that were able to block the binding of the huCD4mIgG recombinant protein to MOLT-BaL cells. The antibodies tested included CD4bs Abs (VRC01, VRC03), anti-glycosylated epitopes (2G12, PGT126), anti-glycosylated-quaternary epitopes (PG9, PG16), CD4 induced epitopes (17b,48d), anti-gp41 (2F5, 4E10) and a polyclonal goat anti-gp120 (anti-gp120) obtained by immunization using rgp120.</p

Characteristic of ART-naive HIV-1 infected patients.

<p>Characteristic of ART-naive HIV-1 infected patients.</p

Detection of CD4bs antibodies by ELISA.

<p>The presence of CD4bs antibodies in ART-naive HIV-1 infected patients (HIV-1) was evaluated by ELISA using the RSC3 and the RSC3Δ371I (RSC3Δ) recombinant proteins. A) Standard curve was calculated using the 2G12 antibody, which recognized both recombinant proteins. The antibody IgGb12, which recognized RSC3 but not RSC3Δ, was used as control. B) The titer of CD4bs antibodies was calculated as RSC3-RSC3Δ. Differences with uninfected healthy donor controls (HC) were evaluated by Fisher´s exact test. Dotted line show cut-off of positivity calculated as mean+2xSD of HC samples. C) Levels of CD4bs antibodies were determined in two plasma samples separated by one year from the same HIV-1 infected individuals. Dotted line indicated the cut-off as in panel B. No difference between both time points was observed (paired t-test).</p

Comprehensive Antigenic Map of a Cleaved Soluble HIV-1 Envelope Trimer

<div><p>The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.</p></div

3D modeling to explain bNAb competition patterns.

<p>(<b>A</b>) 3D models for the nonreciprocal competition between PG9 and PGT122. Side and top views of the bNAbs are shown together with the footprints. (<b>B</b>) Analysis of the Asn332 supersite depicting the angles of approach to the Asn332 glycan taken by various bNAb subfamilies. (<b>C</b>) 3D models to explain the bidirectional competition between PGT151 and 8ANC195. (<b>D</b>) 3D models to explain the unidirectional competition between OD-glycan and CD4bs bNAbs and (<b>E</b>) 3D models to explain the nonreciprocal competition between 8ANC195 and CD4bs bNAbs.</p

Comparison of PGT135 and VRC01 binding to HIV-1 gp120.

<p><b>(A)</b> View down the trimer three-fold axis showing a superimposition of PGT135 Fab in complex with the clade B JRFL gp120 core (PDB ID: 4jm2; red), and of VRC01 Fab in complex with the clade A/E 93TH057 gp120 core (PDB ID: 3ngb; blue), each aligned onto the crystal structure of the BG505 SOSIP.664 trimer (PDB ID: 4tvp; white). In addition, the same BG505 SOSIP.664 crystal structure is fit into the negative-stain EM reconstruction of JR-FL Env trimer in complex with PGT151 (EMD-5919; white). The resulting model clearly shows that PGT135 and VRC01 do not sterically block binding of each other. <b>(B)</b> Side view of <b>(A)</b> with only one gp120 monomer displayed for clarity. <b>(C)</b> Detailed view of the key glycans (spheres) that were resolved in the PGT135-gp120 structure, and of the same glycans from the BG505 SOSIP.664 crystal structure. The presumed steric clash between glycan on Asn362 (and perhaps also Asn363) and the CDR H2 loop of VRC01 is marked with an asterisk. The gp120 subunit of the BG505 SOSIP.664 trimer is displayed as white ribbons. The glycans depicted were limited to the components resolved in the crystal structures (GlncNAc₁ for Asn362, GlncNAc₂Man₁ for Asn386 and GlncNAc₂Man₆ for Asn392 in the PGT135-gp120 structure; GlncNAc₂ for Asn362, GlncNAc₂ for Asn386 and GlncNAc₂ for Asn392 in the BG505 SOSIP.664 trimer structure), and hence their sizes are underestimated compared to trimers produced in 293F cells or present on viruses.</p

CD4 binding to the BG505 SOSIP.664 trimer and the induction of CD4-like conformational changes.

<p>(<b>A</b>) Use of sCD4 as a competitor. (<b>B</b>) Use of CD4-IgG2 as the analyte. (<b>C</b>) Induction of conformational changes measured by 17b binding. The extent of 17b binding in the presence of sCD4 was defined as 100%. *** indicates a significant difference between 17b binding without sCD4 and in the presence of a bNAb, as calculated using a Mann-Whitney 2-tailed test (P <0.05).</p

bNAb epitopes mapped onto the 3D structure of the BG505 SOSIP.664 trimer.

<p>(<b>A</b>) Various bNAbs (not labeled) to different epitope clusters are modeled onto each protomer of the trimer, according to fitting of various EM density maps (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004767#sec013" target="_blank">material and methods</a>). (<b>B</b>) bNAbs to different epitope clusters are modeled onto the same EM density map. Only one Fab fragment per trimer is shown for clarity. (<b>C</b>) Footprints of the different bNAb Fab fragment densities displayed in (<b>B</b>). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004767#sec013" target="_blank">methods</a> for further details.</p

Cross-competition analysis for bNAb binding to BG505 SOSIP.664 trimer by SPR.

<p>(<b>A</b>) Competition between 8ANC195 (competitor) and PGT151 and 35O22. Association-dissociation curves of the individual binding experiments were overlaid with the second association phase to detect competition. 0 on the y axis is the baseline for the single comparator injection and for the same analyte as the second in the double injection. Thus, the strength of all three responses can be read on the same scale, although the value for the first analyte in the double injection will be negative. (<b>B</b>) Competition between PGT151 (competitor) and 8ANC195 and 35O22. (<b>C</b>) Competition between 35O22 (competitor) and 8ANC195 and PGT151.</p