Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential

Abstract

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

FIG. 1.

Alignment of all of the SU5 sequences analyzed in this study. SU5 sequences deposited in GenBank (underlined) were aligned with newly obtained SU5 sequences (not underlined). Dashed underlines indicate sequences clustering in the MVV group, while continuous underlines mark sequences of the CAEV group. Virus sequences obtained from goats are marked with a bold G, while those obtained from sheep are marked with a bold S.

FIG. 2.

Kinetics of seroconversion in an SU5-total ELISA of goats experimentally infected with SRLV. (A) Results of goat 9614 infected with strain 1355. Sera were tested at different time points (black squares) in a 1355-SU5-total ELISA. Goat 9605 (open diamonds) did not seroconvert and served as a negative control. (B and C) Results of goats 01, 03, and 04 (B) and 10, 11, and 13 (C), respectively. Sera were tested at different time points in a CO-SU5-total ELISA. OD at 405 nm was measured at 30 min after addition of the chromophore. The cutoff (dotted line) was set at an OD of 0.3.

FIG. 3.

Kinetics of antibody avidity maturation. Maturation of the avidity of anti-SU5 antibody from goats 01, 04, 11, and 13 taken at 5, 92, and 208 weeks post experimental infection is shown. The avidity index was calculated as described in Materials and Methods. Serum antibodies with avidity index values of <30% are designated low-avidity antibodies (below the solid line), those with values between 30 and 50% are intermediate-avidity antibodies (between the solid and dotted lines), and those with values of >50% are considered high-avidity antibodies (above the dotted line).

FIG. 4.

Competition for 1163-SU5 peptide binding. Serum A (black column) and serum B (shaded column) were allowed to react with 1163-SU5-total peptides in an ELISA without (no) or with the following competing peptides: 1163-SU5-total, 1163-SU5-variable, 615-SU5-total, 615-SU5-variable, Zn-SU5-total, and Zn-SU5-variable (Table 1). OD at 405 nm was measured at 30 min after addition of the chromophore. The cutoff (dotted line) was set at an OD of 0.3.

FIG. 5.

Analysis of SU5 cross-reacting antibody. Antibodies cross-reacting between phylogenetically distinct SU5 peptides were purified by affinity chromatography from the serum of goat 13, which was experimentally infected with the molecular clone CAEV-CO, on a column prepared with an SU5-total peptide derived from the sequence of distantly related strain 1163. The histogram shows the absorption (OD) measured in an ELISA with the 1163-SU5-total peptide (black columns) or the CO-SU5-total peptide (open columns). All sera were incubated for 30 min in duplicate and subsequently transferred to the next wells. This operation was repeated 11 times (steps) on the matching peptide (1163- or CO-SU5-total, respectively) and resulted in the depletion of peptide-specific reactivity. In the last step (step 12), the sera depleted of CO-SU5-total specific antibody were tested in an 1163-SU5-total ELISA (black column). Conversely, sera depleted of 1163-SU5-total specificity were tested in a CO-SU5-total ELISA (white column). The avidities of the sera marked with thick black lines above the histogram columns were determined. The symbols *, #, ▴, and ▵ indicate the measured avidities to specific samples or sample groups.

FIG. 6.

Serological results of the diagnostic-case sera tested in an SU5-total ELISA. The peptides used were 1355-SU5-total (dark gray), 1163-SU5-total (black), and 615-SU5-total (white). The OD at 405 nm was measured at 30 min after addition of the chromophore. The black line indicates the cutoff value, which was set at an OD of 0.3. G, goat; S, sheep.

FIG. 7.

Phylogenetic analysis of the SU5 sequences obtained from the diagnostic-case isolates. The SU5 regions of the SRLV strains infecting goats 1 and 3 (G1 and G3) and sheep 1 and 4 (S1 and S4) were amplified by PCR and sequenced. The MVV-like group is shown by dash lines, and the CAEV-like group is shown by solid lines. Novel sequences are boxed.