Figure 1.
V2-related Reagents and Assays Used in the Pilot and Case-Control Analyses.
The superscripts denote the following: a “Cyclic V2 (AAs 157–198)” was used in assays in three labs as shown. Throughout this table, blue V2 sequences are identical to the subtype E 92TH023 used in the prime; gray represent scrambled sequences or linkers; brown represents the sequence the central AAs in an extremely polar V2 in subtype A strain QB585.2102M.Ev1v5.C (http://www.hiv.lanl.gov) with individual mutations shown in cyan; black represents sequences chosen for particular properties, as described; green represents the V1V2 from subtype B Case A2 [20] and the central 23-mer of V2 from subtype B strain MN. b All peptides were biotinylated at the N-terminus with the exception of peptide K178 and peptide V2 A244-92TH023 which were biotinylated at the C-terminus. * Multiple V2 peptides from various strains (see text).
Table 1.
Response rate and odds ratios (ORs) calculated from the case-control specimens tested with 13 V2 variables.
Figure 2.
ELISA reactivity of human anti-V2 mAbs 697-D and 2158 with AIDSVAX subtype E (A244, green square) and AIDSVAX subtype B (MN, O) gp120 immunogens.
The pink dashed line represents twice background levels.
Figure 3.
ELISA reactivity with gp70-V1V2 of plasma specimens used in the pilot studies of the RV144 clinical vaccine trial (Set C).
The results from one of three experiments are shown. The open and filled blue diamonds depict negative responses at weeks 0 and 26, respectively. The open and closed red circles depict positive responses at weeks 0 and 26, respectively. Each vertical line connects a single patient's specimen drawn at Week 0 and Week 26. The specimens are ordered by the difference in reactivity between the Week 0 and Week 26 specimens, with the biggest increasers on the right. Plasma were tested at a final dilution of 1∶100, and a positive response was defined as being >0.283, the cut-off OD value which was defined as the mean +3 standard deviations based on values derived from vaccinees at week 0 (the pre-immunization time point).
Figure 4.
Spearman rank correlations between the V2 assays run in the case-control study.
Figure 5.
Boxplots showing ELISA reactivity of V2 peptides with plasma (diluted 1∶100) from 80 vaccinees' specimens from the pilot study (Set C).
The distributions of the reactivities are shown where the left edge of each box equals the 25th percentile; the vertical line in each box is the 50th percentile, and the right edge of each box equals the 75th percentile. The boxplots were prepared using Prism, with “whiskers” showing the minimum and maximum responses. Panel A: Four 21-mer N-terminal biotinylated peptides (Peptides 1–4) were selected on the basis of a bioinformatics analysis of V2 sequences from the LANL HIV Database (see text). Panel B: A second peptide panel designed upon inspection of the AAs in Peptides 1–4 in Panel A revealed AAs that distinguish Peptides 1 and 3 from 2 and 4; these appear at positions 165, 169, 172, and 174. To maximally enhance the availability of the epitopes on the peptides used in the fine mapping of the V2 Abs, a spacer of three glycines was inserted between the biotin tag at the N-terminus of the peptide and the V2 sequences.
Figure 6.
Ribbon diagram of the backbone fold of the V1V2 domain (purple and yellow ribbons) bound to the CDR H3 region of mAb PG9 (transparent stippled spheres are the atoms of the PG9 CDR H3, blue = nitrogen, red = oxygen, white = carbon).
The ribbon backbone of AAs 165–176, which are identical to Peptide 6 (in Figure 5), is colored yellow. The strands are labeled A–D according to the convention established recently [28].
Figure 7.
Estimated odds ratios (OR) and 95% confidence intervals for each of the V2 assays.
Data are derived from the categorical analyses shown in Table 1. Estimated ORs compare subgroups defined by High vs. Low responses except for comparisons for analyses of IgA V2 A244 K178 and V2 MN which compare Positive vs. Negative responses. For evaluation of biotinylated V2 Peptide 6, comparison is between High and Negative responses.
Figure 8.
Hotspot analysis of the microarray data of V2 Abs in the plasma from vaccinees in the case-control study.
The upper panel shows an aggregate response across all sub-types averaged across vaccinees as a function of the V2 sequence in subtype B strain HxB2. An individual aggregate response is computed using a sliding window mean statistic of 9 AAs, i.e., peptides with HxB2 positions of 9 contiguous AAs averaged together. In the lower portion of the figure, the actual sequence of each of the overlapping 15-mers spanning V2 positions 160–183 is shown. The seven sets of peptides represent the consensus V2 regions of HIV-1 Group M and of subtypes A, B, C, D, CRF01_AE and CRF02_AG. Peptides are colored according to their average response across all vaccinees, with a scale of 0 (white) to 1.76 (red). All responses are calculated using normalized intensities and by subtracting the intensities of baseline pre-bleeds.