Antigenic Site-Specific Competitive Antibody Responses to the Fusion Protein of Respiratory Syncytial Virus Were Associated With Viral Clearance in Hematopoietic Cell Transplantation Adults

Abstract

Background: Recent studies of human sera showed that the majority of the respiratory syncytial virus (RSV) neutralizing antibodies are directed against pre-fusion conformation of the fusion (F) protein of RSV and revealed the importance of pre-fusion antigenic site Ø specific antibodies. However, detailed analysis of multiple antigenic site-specific competitive antibody responses to RSV F protein and their contribution to virus clearance in humans are lacking. Methods: We prospectively enrolled a cohort of RSV infected hematopoietic cell transplantation (HCT) adults (n = 40). Serum samples were collected at enrollment (acute, n = 40) and 14 to 60 days post-enrollment (convalescent, n = 40). Antigenic site-specific F protein antibodies were measured against pre-fusion site Ø, post-fusion site I, and sites II and IV present in both the pre-fusion and post-fusion F protein conformations utilizing four different competitive antibody assays developed with biotinylated monoclonal antibodies (mAb) D25, 131-2A, palivizumab, and 101F, respectively. The lower limit of detection were 7.8 and 1.0 μg/mL for the competitive antibody assays that measured site Ø specific response, as well as sites I, II, and IV specific responses, respectively. Neutralizing antibody titers to RSV A and B subgroups was determined by microneutralization assays. Results: The overall findings in RSV infected HCT adults revealed: (1) a significant increase in antigenic site-specific competitive antibodies in convalescent sera except for site Ø competitive antibody (p < 0.01); (2) comparable concentrations in the acute and convalescent serum samples of antigenic site-specific competitive antibodies between RSV/A and RSV/B infected HCT adults (p > 0.05); (3) significantly increased concentrations of the antigenic site-specific competitive antibodies in HCT adults who had genomic RSV detected in the upper respiratory tract for <14 days compared to those for ≥14 days (p < 0.01); and (4) statistically significant correlation between the antigenic site-specific competitive antibody concentrations and neutralizing antibody titers against RSV/A and RSV/B (r ranged from 0.33 to 0.83 for acute sera, and 0.50-0.88 for convalescent sera; p < 0.05). Conclusions: In RSV infected HCT adults, antigenic site-specific antibody responses were induced against multiple antigenic sites found in both the pre-fusion and post-fusion F conformations, and were associated with a more rapid viral clearance and neutralizing antibody activity. However, the association is not necessarily the cause and the consequence.

Keywords: competitive antibody; fusion protein; hematopoietic cell transplantation; respiratory syncytial virus; viral clearance.

Figure 1

Specificity of RSV antigenic site-specific competitive antibody assays. Blue indicates there was the mAbs had no inhibition to the biotinylated mAbs specific to the RSV antigenic site. Red indicates there were strong inhibition to the RSV antigenic site. The 0.0–1.5 scale represents the level of mAb inhibition from the strongest to the weakest. PVZ, palivizumab; MVA, motavizumab. Data represented are from 3 independent duplicate competitive antibody assays.

Figure 2

Neutralizing potency of RSV antigenic site-specific monoclonal antibody. RSV/A Tracy and RSV/B 18537 were used in the experiments. The concentration of the mAbs were 40 μg/mL for D25, palivizumab, 131-2A, and 101F in both RSV/A and RSV/B microneutralization assays, except that mAb 131-2A was used at 1.0 mg/mL in the RSV/B microneutralization assay. The Y axis is the RSV/A Tracy or RSV/B 18537 neutralizing antibody titers (log2) for different mAbs used in the microneutralization assay. Data represented are from 3 independent duplicate microneutralization assays ± standard deviation.

Figure 3

Percentage of each RSV antigenic site-specific competitive antibody GMC (μg/mL) to the total GMC (μg/mL) for acute and convalescent sera from HCT adults shedding virus <14 days and ≥14 days. The Y axis is the percentage of each competitive antibody GMC to the total GMC. The X axis is the serum types: acute or convalescent sera from HCT adults shedding virus <14 days or ≥14 days. The total GMC was the sum of 4 competitive antibody GMC. The percentage of each competitive antibody was that the competitive antibody GMC divided by the total GMC. GMC, geometric mean concentration. N = 40 in each competitive antibody assay.

Figure 4

Percentage of each RSV site-specific competitive antibody GMC (μg/mL) to the total GMC (μg/mL) for RSV/A and B infected HCT adults. GMC, geometric mean concentration. n = 40 in each competitive antibody assay.

Figure 5

Correlation of RSV antigenic site-specific competitive antibody and RSV-specific neutralizing antibody. Pearson's correlation coefficient was calculated to measure the strength of the linear association. Correlation coefficients ranged from 0.33 to 0.83 for acute sera, and 0.50–0.88 for convalescent sera. ^*Correlation is significant at the 0.05 level (2-tailed). ^**Correlation is significant at the 0.01 level (2-tailed). n = 40 in each competitive antibody assay and microneutralization assay.