Article Figures & Data
Figures
- FIG. 1.
(A) Neutralization curves for a panel of 28 sera (from HIV-1-infected humans) against SF162 (top panel) and JR-FL (bottom panel) pseudotypes. Sera selected for high-level anti-gp120 titers were serially diluted and preincubated for 30 min with pNL4-3 luc virions pseudotyped with either SF162 or JR-FL Env proteins; the mixture was then added to 96-well plates containing U87/CD4/CCR5 cells. Luciferase activity was measured after 3 days, and percent neutralization was calculated by dividing the difference between that value and luciferase activity for a control sample (virus without antibody) and multiplying by 100. (B) Scatter graph showing relationship between ND50s obtained for SF162 pseudotypes versus those for JR-FL pseudotypes for individual sera analyzed as described for Fig. 1A. The best-fit regression line and r2 value are shown.
- FIG. 2.
Representative neutralization patterns for four human sera (serum samples 2, 4, 16, and 25) with high-level neutralization selectivity for SF162 against pNL4-3 luc virions pseudotyped with SF162 env or JR-FL env.
- FIG. 3.
(A) Binding curves of anti-V3 MAbs to rgp120 derived from SF162 and JR-FL env genes. The soluble gp120s were captured with sheep anti-C5 polyclonal antibody; antibody binding was determined at the indicated time points as described in Materials and Methods. (B) Neutralization of SF162 and JR-FL HIV pseudotypes by selected anti-V3 MAbs. Neutralization assays were performed with U87 cells expressing CD4 and CCR5 receptors; percent neutralization was determined as described above. OD 405 @ 20′, optical density at 405 nm after 20 min; conc, concentration.
- FIG. 4.
(A) Binding curves of anti-CD4-bs MAbs to SF162 (squares) and JR-FL (triangles) rgp120s. Binding assays were performed as described for the anti-V3 MAbs. (B) Neutralization curves of three anti-CD4-bs MAbs against SF162 (squares) and JR-FL (triangles) pseudotypes of pNL4-3 luc. OD 405, optical density at 405 nm; conc, concentration.
- FIG. 5.
Sequence alignment of the V1/V2 domains of SF162 and JR-FL gp120 proteins. Homologous residues are indicated by hyphens; modified asparagines of N-linked glycosylation sites are marked with asterisks. Numbering is based on the relative positions in the HXB2 sequence.
- FIG. 6.
Representative neutralization curves for human sera against SF162 and JR-FL parental and JR(SF-V1/V2) and SF(JR-V1/V2) Env pseudotypes. The three left panels show patterns for sera that preferentially neutralize SF162 pseudotypes; the three right panels show patterns for the sera with the greatest cross-neutralizing activities.
- FIG. 7.
Representative neutralization curves for MAbs against SF162 and JR-FL parental and JR(SF-V1/V2) and SF(JR-V1/V2) Env pseudotypes.
Tables
- TABLE 1.
Ratios of 50% maximal binding concentrations and ND50s of MAbs to SF162 and JR-FL pseudotypes
Epitope MAb 50% Maximal binding concna Binding ratio (JR/SF) ND50b ND50 ratio (JR/SF) JR-FL SF162 JR-FL SF162 V3 447-52D 0.009 0.009 1.0 15 0.015 1,000 4117C 0.026 0.023 1.1 35 0.015 2,333 4148D 0.14 0.13 1.1 100 0.042 2,381 2191 0.024 0.021 1.1 18 0.004 4,500 2219 0.017 0.021 0.8 24 0.003 8,000 2182 0.0024 0.01 0.2 33 1.1 30 CD4-bs 5145A 0.081 0.057 1.4 50 0.040 1,250 1125H 0.29 0.11 2.6 >>100 1 >>100 IgG-b12 0.035 0.040 0.9 0.037 0.032 1.2 V2 8.22.2 0.51 0.56 0.9 >>100 37 >>3 2158 0.067 (1.3)c (0.05)c >>50 11 >>5 830A 0.04 (0.5)c (0.08)c >50 2 >25 CD4-i X5 (Fab) NDe ND ((20))d 47 0.54 87 17b ND ND ((2.4))d >>100 16 >>6 48d ND ND ND >>100 4.8 >>20 2G12 2G12 0.12 0.42 0.3 0.38 2.4 0.16 gp41 2F5 NAf NA NA 0.38 1.3 0.29 ↵ a Titers were determined for serially diluted MAbs in ELISA plates coated with purified gp120 antigens; antibody concentrations (in micrograms per milliliter) that gave 50% maximal binding are reported.
↵ b ND50s were determined for pNL4-3 luc virions pseudotyped with the indicated envelope proteins by interpolation from neutralization curves and are reported in micrograms per milliliter.
↵ c The 50% maximal binding concentrations and ratios listed in parentheses represent values estimated by assuming that the maximum binding plateau for SF162 gp120 (value not achieved) was the same as that for JR-FL gp120.
↵ d Binding plateaus were not obtained. Values listed in double parentheses represent ratios of concentrations required to achieve an optical density of 0.55 after 25 min.
↵ e ND, not determined (binding plateaus not obtained).
↵ f NA, not applicable.
- TABLE 2.
Relative ND50s of human sera for parental and chimeric SF162/JR-FL Env pseudotypes
Human serum ND50 for pseudotypea: SF162 JR(SF V1/V2) JR-FL SF(JR V1/V2) 2 90,000 115,000 <20 <20 4 43,000 75,000 <20 120 25 31,000 13,000 <20 <20 16 25,000 16,000 <20 <20 04-2594k 14,000 10,800 <20 <20 26 14,000 10,500 <20 40 04-2435j 13,500 8,500 <20 <20 04-1642F 12,800 32,000 <20 <20 04-8942K 12,200 13,500 <20 20 9 11,000 11,800 <20 20 04-2416d 9,000 13,600 <20 <20 6 51,000 36,000 130 1,500 04-1360c 27,000 36,000 95 140 04-3058c 10,000 16,800 60 270 3 11,500 12,800 80 140 04-3062c 9,500 8,700 90 150 17 12,700 9,100 140 1,480 04-1657F 71,000 66,000 930 5,700 04-2132E 75,000 93,000 1,300 3,000 21 10,500 29,000 180 1,100 04-0748D 48,000 48,000 1,150 5,500 10 6,500 13,000 160 135 04-2213c 6,700 12,000 180 1,000 22 10,000 14,000 400 1,000 04-0405J 90,000 120,000 4,000 700 04-03552A 12,800 97,000 1,400 5,300 18a 7,500 29,000 1,100 2,900 04-2583b 52,000 78,000 12,000 58,000 NHSb <20 <20 <20 <20 - TABLE 3.
ND50s of MAbs for SF162 and JR-FL parental and chimeric viral pseudotypes
Epitope MAb ND50 ND50 for pseudotypea: SF162 JR(SF V1/V2) JR-FL SF(JR V1/V2) V3 447-52D 0.015 0.0013 15 >50 4117C 0.015 0.005 35 >50 4148D 0.042 <0.01 100 >100 2191 0.004 0.002 18 >5 2219 0.003 0.002 24 >5 2182 1.1 0.002 33 >50 V2 8.22.2 37 20 >>100 >>100 830A 2 2 >50 >50 2158 11 10 >>50 >>50 CD4-bs IgG-b12 0.032 0.04 0.037 0.025 5145A 0.04 0.18 50 50 1125H 1 0.8 >>100 >>100 sCD4 0.41 0.1 3.7 >10 gp41 2F5 1.3 0.26 0.38 3.4 CD4i 17b 16 6.5 >>100 >100 48d 4.8 5.2 >>100 >>100 X5 0.54 0.25 47 19 Other 2G12 2.4 0.20 0.38 2.5 ↵ a Concentration of antibody (in micrograms per milliliter) required to reduce luciferase activity by 50%.
