Genetic diversity of HIV-1 gp140s present in immunogen Mixes 1–6.

<p>The phylogeny was constructed from the <i>env</i> nucleotide sequence alignment using a maximum likelihood model. Edges are coloured according to mix. Due to a high representation of expressing subtype B Envs, the 19 subtype B Envs were partitioned into 2 subtype B groups designated B and B′.</p

List of selected immunogens.

<p>Bx08 was used as benchmark in a pilot experiment.</p>a<p>length: number of amino acids.</p>b<p>PNGS: number of potential N-linked glycosilation sites as identified using N-glycosite at the HIV database website (<a href="http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html" target="_blank">http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html</a>).</p

Immunization schedule.

<p>(<b>A</b>) Timeline of DNA and protein immunizations. Macaques were each given two DNA priming injections spaced by two months followed by a four-month interval and then three gp140 protein boosts, spaced by two-month intervals. Blood was collected at the time of each protein boost and two weeks after each boost (*). A final bleed was collected eight weeks after the final boost. (<b>B</b>) Each immunization consisted of six separate injections at different sites on the macaques. In the heterotrimer group the six different immunogens were rotated around these sites so that any local draining lymph nodes experienced different immunogens after each injection. Details given here indicate which Mix immunogen was administered at each site (1–6) and the contents of those mixes; ‘15 × subtype A’ indicates that Mix 1 contains heterotrimers generated from 15 subtype A Envs.</p

Kinetics of neutralizing responses against SF162 pseudovirus.

<p>Kinetics using three 2-fold dilutions of IgG isolated from weeks 4, 8, 12 and 14 sera of rabbits immunized with trimer ITM1_4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074552#pone-0074552-g006" target="_blank">Figure 6a</a>), ACS19554 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074552#pone-0074552-g006" target="_blank">Figure 6b</a>) and ACS19642 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074552#pone-0074552-g006" target="_blank">Figure 6c</a>). Horizontal lines indicate the mean percent neutralization. Final IgG concentrations were 250 µg/ml (filled dots), 125 µg/ml (semi filled dots) and 62.5 µg/ml (open circles) respectively. The dotted (green) line represents 50% neutralization.</p

Peptide inhibition experiments.

<p>Four serial 2-fold dilutions of IgG purified from rabbits immunized with ITM1_4 (ITM1_4-1; -2; -3 and -4) and UG_A (UG_A-1; -2; -3 and -4) were used in a modified pseudovirus neutralization assay using SF162. Two linear peptides 797 and 7012.1, recognizing the V3-crown motif of clade B (GPGR) and clade A (GPGQ) respectively and 1 cyclic peptide 7019 recognizing GPGR (clade B) were used at a final concentration of 16 µg/ml. Each graph represents a single rabbit.</p

Immunization in the presence or absence of CAF01.

<p>(A) End-point binding titer of rabbits immunized with ITM1_4, in the presence (filled triangles) or absence (open triangles) of CAF01 respectively. Results are given for weeks 8, 12 and 14. Each dot represents one rabbit. Horizontal lines indicate mean titers. Statistical analysis was done using Mann-Whitney test. (B) Neutralization using IgG (at a final concentration of 250 µg/ml) isolated from week 12 (filled squares) and 14 (open triangles) sera of rabbits immunized with ITM1_4 in the presence or absence of CAF01. Two clade B viruses, SF162 (left) and Bx08 (right) were used. Horizontal lines indicate the mean percent neutralization. The dotted (green) line represents 50% neutralization. Statistical analysis was done using Mann-Whitney test.</p

Comparison of trimeric gp140 versus monomeric gp120.

<p>(A) End-point binding titers of rabbits immunized with either trimeric gp140 (filled squares) or monomeric gp120 (open squares) of UG_A HIV-1 Env. Each dot represents one rabbit. Horizontal lines indicate mean titers. Statistical analysis was done using Mann-Whitney test. (B) Neutralization data using IgG (at a final concentration of 250 µg/ml) isolated from week 12 sera of rabbits immunized, in the presence of CAF01, with either trimeric gp140 or monomeric gp120 of UG_A. Neutralization of two clade B viruses, SF162 (left, squares) and Bx08 (right, diamonds) is depicted. Each dot represents one rabbit. Horizontal lines indicate the mean percent neutralization. Statistical analysis was done using Mann-Whitney test.</p

ID₅₀ tier-1 pseudotyped virus neutralization titers in the TZM-bl assay.

#<p>Numbers given are the sample dilutions at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. All samples were taken two weeks after the third protein boost.</p>$<p>Values above the median titer for each pseudovirus are shown in bold.</p><p>ID₅₀ tier-1 pseudotyped virus neutralization titers in the TZM-bl assay.</p

ID₅₀ tier-2 pseudotyped virus neutralization titers in the A3R5 assay.

#<p>Numbers given are the sample dilutions at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. All samples were taken two weeks after the third protein boost.</p>$<p>Values above the median titer for each pseudovirus are shown in bold.</p><p>ID₅₀ tier-2 pseudotyped virus neutralization titers in the A3R5 assay.</p

Cross-competition ELISA.

<p>Titration series of sera from two weeks after the third protein boost were competed with biotinylated mAbs or biotinylated soluble domains 1–4 monomeric CD4 using UG37 gp140 as the antigen and 50% inhibition titers (IT50) were calculated as the serum dilution at which binding of the biotinylated probes was reduced by 50% compared to control well without serum. Epitopes targeted by the mAbs are specified in parentheses. CD4bs: CD4 binding site; CD4i: CD4 inducible epitope. A one-way ANOVA was performed for each competing protein/antibody with Dunnett’s post-hoc tests to compare Group 1 and Group 3 with the control Group 2. Significance is illustrated by **p<0.001.</p