Peak reciprocal anti-trimer Ab end-point titres in rabbit sera, ranked by immunization group.

<p>Peak reciprocal anti-trimer Ab end-point titres in rabbit sera, ranked by immunization group.</p

Expression of BG505s trimers in tissue culture.

<p>(A) Monolayers of HEK 293T cells were infected with ChAdOx1.BG505s at the indicated MOIs for 48 h. The proteins in the tissue culture supernatants were separated by non-denaturing polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. BG505 Env proteins were detected using the glycan-specific 2G12 mAb as the primary antibody. As a positive control, supernatant from a HEK 293T stable cell line that expresses fully cleaved BG505s trimers was analysed in the first lane. The relative molecular masses of 600 and 450 kDa are indicated. (B) To assess the percentage of correctly folded trimers in the ChAdOx1.BG505s- and MVA.BG505s-infected cell culture supernatants, the ELISA plates were first coated with either 2G12 or PGT151 Env-specific bnAbs and the captured native-like, furin-cleaved trimers were detected using PGT145 as the secondary mAb. To generate the standard curve used for trimer quantification (top), gp140 monomers and trimers purified from stably transfected cells were mixed at different ratios, but at a constant total concentration. The latter values were determined using a different, non-trimer specific capture ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181886#sec002" target="_blank">Methods</a> section). The percentages of native-like BG505s trimer present in the virus-infected cell culture supernatants are shown for the indicated antibody combinations (bottom).</p

Antibody recognition of glycan holes^a.

<p>Antibody recognition of glycan holes<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181886#t003fn001" target="_blank">^a</a>.</p

Neutralization of tier-1 and tier-2 viruses in TZM-bl cell assay^a.

<p>Neutralization of tier-1 and tier-2 viruses in TZM-bl cell assay<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181886#t003fn001" target="_blank">^a</a>.</p

Kinetics of BG505s trimer-binding antibody responses induced in rabbits by various immunization regimens.

<p>(A) Groups of 5 rabbits were immunized with ChAdOx1.BG505s (C), MVA.BG505s (P) and ISCOMATRIX^™-adjuvanted BG505s protein trimer (P) candidate vaccines, as depicted. The animals were immunized at weeks 0, 8 and 24 (arrows), and bled regularly. (B) Reciprocal endpoint titres of the BG505s trimer-binding antibodies in rabbit sera were determined by capture ELISA. The trimer-binding antibody titres at each time point are shown for individual rabbits, and the median values for group-peak time points are shown above each peak. Note that the time points at which the peak titres were measured differed among the rabbits in each group, and that some rabbits died for protocol-unrelated reasons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181886#pone.0181886.s001" target="_blank">S1 Table</a>).</p

Endpoint IgG binding titer.

<p>The antigen-specific IgG titer was measured by ELISA. Antiserum (terminal time point) was serially diluted (2-fold) and the endpoint titer was defined as the last dilution that gave a positive signal (>3-fold signal of the prebleeds). Animals that received the FCA/FIA (groups 1 and 2) or combination of Carbopol-971P and MF59 have significantly higher endpoint IgG titers than others (p<0.05).</p

Monoclonal antibody binding to gp140_SF162 in the presence of adjuvant.

<p>The antigenicity of gp140_SF162 in the presence of adjuvants was measured by mAb binding. A panel of 12 different mAbs, representing different epitope domains of the Env, was used. The signal of mAb binding to the adjuvant-formulated gp140_SF162 was calculated relative to the signal measured in the unadjuvanted sample, which is assigned to have the value of 1. The data is color coded: Yellow represents no difference in mAb binding, or no change of antigenicity as compared to the unadjuvanted sample and light blue represents up to 3-fold decrease of mAb binding (or antigenicity). Both FIA and MF59 adjuvants appeared to have little effect on antigenicity, as mAb binding was very similar to the unadjuvanted sample. On the other hand, decreased mAb bindings were detected in the presence of both of the Carbopols.</p

Relative avidity index.

<p>Avidity of the antiserum was measured by its ability to remain in binding with gp140_SF162 in the presence of 8 M Urea. All samples were found to have relative high avidity, defined as >50% IgG binding in the presence of 8 M Urea, as compared to the no Urea control. Compared to the unadjuvanted group (no. 12), antisera from animals vaccinated with adjuvants have significantly higher RAI (p<0.05).</p

gp140_SF162 conformation upon formulation with adjuvant.

<p>The gp140_SF162 glycoprotein was formulated with or without adjuvants and assayed on PAGE gel for conformation. (a) Regardless of presence or absence of adjuvants, a single band at 140 kDa was observed in the reduced gel. (b) Similar observation was made in the non-reduced gel, where all the samples showed two bands at 140 kDa and 280 kDa, representing the non-crosslinked and disulfide crosslinked gp140 trimers, respectively. (c) A BN-PAGE gel was used to examine the native conformation of gp140 in the presence of adjuvants. None of the adjuvants appear to affect the structure of gp140.</p

Mean inhibitory concentration (IC) 50 values for duplicate assays performed with TriMab and virus as indicated in the NeutNet Phase I (P1) and Phase II (P2) study.

<p>The cells are colour coded: green, poor or no neutralization, IC50>25 µg/ml; yellow, IC50 5–25 µg/ml; orange, IC50 1–5 µg/ml; red, IC50<1 µg/ml; white, no results available. Assays are grouped on the basis of several criteria: (1) the use of plasmids or culture supernatants as a source of HIV-1; (2) fusion based assays or infection based assays, either with pseudotyped virus or replication competent virus; and (3) the use of cell lines or PBMC. Laboratories performing the assays are numbered (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036438#pone-0036438-g001" target="_blank">Figure 1</a> for reference) and colour coded: blue, TZMbl assay or PSV/plasmid assays; green, PBMC assays using extracellular p24 as readout; pink, plaque reduction assay. In the listing of viruses, to the left, the cells of X4 viruses are labelled grey, the cells of R5 viruses are white.</p