Figure 1.
NAb activities against HIV-1 pseudotyped viruses.
Neutralization was shown as the percent inhibition of virus infection at 1∶20 dilution of the patient sera or using 10 µg/ml of monoclonal antibodies (b12, 2G12, and 4E10). “−“ indicates that percent of neutralization was below 50%. Percent of neutralization between 50–75% or greater than 75% are highlighted in yellow or red, respectively. The HIV-1 pseudotyped viruses used for neutralization assays are shown in the second row from top from “NL4-3” to “CM235.c11” with subtype indicated above the virus panel. “MuLV” is the murine leukemia virus used as negative control for neutralization assays. “NJ0XX”s in the far left column indicates individual patient serum samples; patient HIV subtypes are indicated above each group of samples. Known neutralizing monoclonal antibodies, IgG1 b12, 2G12 and 4E10, served as positive controls for neutralization assays.
Figure 2.
Subtype specificity of NAb responses identified in HIV-1 infected patients’ sera.
Neutralization against pseudotyped viruses expressing primary Env from subtypes B, BC, C, and AE isolates were tested. Percentage of neutralization by patients’ sera (at 1∶20 dilution) infected with one of three viral subtypes (B, BC, and AE as shown in panels A, B, and C, respectively) were calculated. The significant differences (p<0.05) of neutralizing activities between different groups of patient sera are indicated.
Figure 3.
Improvement of NAb responses after one year of HIV-1 infection.
Panels A, B, C and D represent the percent (%) of neutralization at 1∶20 serum dilution against two sensitive subtype B viruses (SF162 and NL4-3), within 1 year, between 1–2 years, longer than 2 years of infection, or time of initial infection unknown. Panels E and F represent the percentage of HIV-1 infected patient sera that showed positive NAb responses (using IC50 at 1∶20 serum dilution as the cut-off) based on length of infection (<1 year, 1–2 years, and >2 years). Panel A shows neutralizing activities against two sensitive viruses (SF162 and NL4-3) and panel B against 44 pseudotyped viruses expressing primary Env antigens (see Fig. 1). The levels of statistical difference between patient sera with different lengths of infection are indicated, which were determined by Fisher exact test.
Figure 4.
Analysis of possible correlations between neutralization breadth in HIV-1 patient sera and selected known neutralizing epitopes V3 and CD4bs.
A. Analysis on the relationship between the reduction of neutralizing activities after V3 peptide absorption in patient sera and the breadth of neutralization with the same patient sera. Percent reduction of neutralizing activities against pseudotyped SF162 was tested with individual patient serum (1∶20 dilution) after absorption with the subtype B consensus V3 peptide. The alignment of subtype B consensus (Cons-B) and SF162 V3 sequence is shown. The number of primary Env pseudotyped viruses (total 44) neutralized by each individual patient serum were calculated as shown in Fig. 1. Correlations and significance were determined using the Spearman method. B. The relationship between antibody titers to compete against neutralizing mAb b12 and the breadth of neutralization (number of viruses being neutralized) in the same patient sera was analyzed. Correlations and significance were determined using the Spearman method.
Figure 5.
MPER peptide inhibition of neutralizing activities of Env-specific mAbs and HIV-1 patient sera.
A: MPER inhibition of NAb activities of mAbs (b12 against CD4bs, 2F5 and 4E10 against MPER) as control; B: MPER inhibition of NAb activities of patient sera with relative broad NAb activities (NJ009, NJ010, NJ012, NJ019, NJ025, NJ028, NJ030, NJ034, and NJ040).
Table 1.
CD4bs mAb competition antibody titers.
Table 2.
Antibody titers competing against Env-specific mAbs 2G12 and PG9 in patient sera with relative broad NAb activities.*