Age-Specific Profiles of Antibody Responses against Respiratory Syncytial Virus Infection

Abstract

Respiratory syncytial virus (RSV) is one of the most prevalent causative agents of lower respiratory tract infections worldwide, especially in infants around 3 to 4months old. Infants at such a young age have maternally-transferred passive antibodies against RSV but do not have active immune systems efficient enough for the control of RSV infection. In order to elucidate age-specific profiles of immune responses against RSV protection, antibody responses were examined by using blood samples in both acute and convalescent phases obtained from child patients and adult patients. In addition to the serum neutralization activity, antibody responses to the RSV fusion protein (F protein) were dissected by analyzing levels of total IgG, IgG subclasses, the binding stability, and the levels of antibody for the neutralization epitopes. It was suggested that children's antibody responses against RSV are matured over months and years in at least 5 stages based on 1) levels of the neutralization titer and IgG3 for F protein in the convalescent phase, 2) geometric mean ratios of the neutralization titers and levels of IgG1 and IgG2 for F protein in the convalescent phase compared to those levels in the acute phase, 3) the affinity maturation of IgG for F protein and the cross reactivity of IgG for RSV glycoproteins of groups A and B, 4) levels of neutralization epitope-specific IgG, and 5) augmentation of overall antibody responses due to repetitive RSV infection.

Keywords: F protein; IgG3; Palivizumab-like neutralization antibody; RSV.

Fig. 1

Serum neutralization activity upon natural RSV infection. (a) Sera were subjected to a microneutralization assay to determine neutralization titers against RSV A2 strain. A left panel represented neutralization titer in acute phase or convalescent phase, and a right panel did the GMRs C/A of NT. The mean ± 95% CI was estimated by linear model, and was shown in the graphs. Neutralization titer in acute phase: 0–3 mo, n = 34; 4–6 mo, n = 30; 7–12 mo, n = 33; 13–18 mo, n = 35; 19–36 mo, n = 35; adults, n = 23. Neutralization titer in convalescent phase: 0–3 mo, n = 35; 4–6 mo, n = 30; 7–12 mo, n = 34; 13–18 mo, n = 35; 19–36 mo, n = 35; adults, n = 23. GMRs C/A of NT: 0–3 mo, n = 34; 4–6 mo, n = 30; 7–12 mo, n = 33; 13–18 mo, n = 35; 19–36 mo, n = 35; adults, n = 23. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (b) Sera, which showed high neutralization activity against RSV A2 in panel (a), were subjected to a microneutralization assay to evaluate NT against RSV 18537 strain. Children: r = 0.705, p < 0.0001 (n = 24), adults: r = 0.657, p = 0.0012 (n = 21). (c) Neutralization titers were also analyzed in two distinct groups according to RSV groups infected during the research period to evaluate the cross neutralization activity at convalescent phase. Group A: 0–3 mo, n = 20; 4–6 mo, n = 16; 7–12 mo, n = 17; 13–18 mo, n = 25; 19–36 mo, n = 21; adults, n = 12. Group B: 0–3 mo, n = 14; 4–6 mo, n = 11; 7–12 mo, n = 13; 13–18 mo, n = 6; 19–36 mo, n = 8; adults, n = 5. The mean ± 95% CI was estimated by the linear model, and were shown in the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 2

Serum antibody response to RSV F protein. The sera were subjected to ELISA coating RSV F protein. Series of detection antibody labeled with HRP were used to acquire the profile of IgG subclasses specific to RSV F protein. A left panel represents concentration of total IgG, IgG1, IgG2 and IgG3 for RSV F in acute phase or convalescent phase, and a right panel does the GMRs C/A of F IgGtotal, IgG1, IgG2, and IgG3. The mean ± 95% CI was estimated by the linear model, and were shown in the graphs. Concentration of antibody in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 28; 7–12 mo, n = 29; 13–18 mo, n = 34; 19–36 mo, n = 35; adults, n = 23. Concentration of antibody in convalescent phase: 0–3 mo, n = 28; 4–6 mo, n = 30; 7–12 mo, n = 33; 13–18 mo, n = 35; 19–36 mo, n = 34; adults, n = 23. GMRs C/A: 0–3 mo, n = 23; 4–6 mo, n = 28; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 34; adults, n = 23. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 3

Evaluation of serum IgG to recognize antigenic sites I, II, and IV in F protein. Sera were subjected to competitive ELISA by using monoclonal antibody specific to antigenic sites I, II, or IV. The amounts of serum IgG specific for each antigenic epitopes are represented by IC50 resulting in 50% reduction of biding RSV F with competitive monoclonal antibody. The limitation of the IC50 detection was defined as 5. The concentration of serum IgG specific to antigenic site II was calculated based on the result of palivizumab on competitive ELISA. The mean ± 95% CI was estimated by the linear model, and were shown in the graphs. IC50 of antigenic site I-specific IgG in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 26; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 32; adults, n = 23. IC50 of antigenic site I-specific IgG in convalescent phase: 0–3 mo, n = 24; 4–6 mo, n = 27; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 32; adults, n = 23. GMRs C/A of antigenic site I-specific IgG: 0–3 mo, n = 21; 4–6 mo, n = 25; 7–12 mo, n = 26; 13–18 mo, n = 34; 19–36 mo, n = 31; adults, n = 23. IC50 of antigenic site II-specific IgG in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 28; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 33; adults, n = 23. IC50 of antigenic site II-specific IgG in convalescent phase: 0–3 mo, n = 27; 4–6 mo, n = 29; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 33; adults, n = 23. GMRs C/A of antigenic site II-specific IgG: 0–3 mo, n = 24; 4–6 mo, n = 27; 7–12 mo, n = 26; 13–18 mo, n = 34; 19–36 mo, n = 32; adults, n = 23. IC50 of antigenic site IV-specific IgG in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 28; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 31; adults, n = 23. IC50 of antigenic site IV-specific IgG in convalescent phase: 0–3 mo, n = 27; 4–6 mo, n = 29; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 33; adults, n = 23. GMRs C/A of antigenic site IV-specific IgG: 0–3 mo, n = 24; 4–6 mo, n = 27; 7–12 mo, n = 26; 13–18 mo, n = 34; 19–36 mo, n = 31; adults, n = 23. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 4

Antibody affinity to RSV F protein. Sera were subjected to a surface plasmon resonance (SPR) assay. Apparent dissociation rate constants (k_d) were calculated and shown in different age groups. The mean ± 95% CI was estimated by linear model, and were shown in the graphs. k_d in acute phase: 0–3 mo, n = 30; 4–6 mo, n = 29; 7–12 mo, n = 30; 13–18 mo, n = 30; 19–36 mo, n = 32; adults, n = 22. k_d in convalescent phase: 0–3 mo, n = 29; 4–6 mo, n = 30; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 33; adults, n = 23. GMRs C/A of F IgG k_d: 0–3 mo, n = 25; 4–6 mo, n = 29; 7–12 mo, n = 28; 13–18 mo, n = 30; 19–36 mo, n = 31; adults, n = 22. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 5

The correlation between the serum neutralization activity and the antibody affinity maturation to F protein. Two-dimensional plots were prepared to investigate the correlation between serum neutralization titer (NT) and k_d. Red arrows represents the changes from acute phase to convalescent phase to show an apparent positive correlation between IgG binding stability and NT, i.e., increase of k_d accompanied with decrease of NT or decrease of k_d with increase of NT. Green arrows represents the changes to show an apparent negative correlation between IgG binding stability and NT. In each age group, regression coefficient of NT on k_d was calculated. 0–3 mo, n = 34; 4–6 mo, n = 30; 7–12 mo, n = 34; 13–18 mo, n = 35; 19–36 mo, n = 34; adults, n = 23.

Fig. 6

Serum antibody response to RSV G protein. The sera were subjected to ELISA coating RSV G protein of group A or group B. Serum IgG concentrations against G of group A and group B in acute phase and convalescent phase were measured, and then GMRs C/A of total IgG specific for G protein were calculated. Data were analyzed in different age groups and in different RSV groups infected during the research period. Plots represent the GMRs C/A of G IgG. 0–3 mo: n = 16 (group A), n = 9 (group B); 4–6 mo: n = 16 (group A), n = 10 (group B); 7–12 mo: n = 16 (group A), n = 9 (group B); 13–18 mo: n = 25 (group A), n = 6 (group B); 19–36 mo: n = 19 (group A), n = 8 (group B).