Evaluation of Immune and Vaccine Competence in Steroid-Sensitive Nephrotic Syndrome Pediatric Patients

Abstract

Idiopathic nephrotic syndrome is a childhood renal disease characterized by a damage of the glomerular filtration barrier leading to an intense leakage of proteins into the urine. This severe proteinuria causes a transient but strong reduction of serum IgG. Therefore, evaluation of vaccine competence by measuring serum levels of protective antibodies can be misleading in nephrotic syndrome, especially during the active phase of disease. To overcome this issue, in parallel to measuring serum antigen-specific IgG, we quantified by ELISPOT the number of antigen-specific memory B cells induced by previous immunization with tetanus and hepatitis B virus (HBV) in 11 steroid-sensitive nephrotic syndrome (SSNS) pediatric patients at onset before any immunosuppressive treatment (mean age 5.1±0.9 years). Five age-matched children with non-immunomediated nephro-urologic disorders were also enrolled as controls (mean age 6.9±2.3 years). Low total serum IgG levels (<520 mg/dl) were found in all the analyzed SSNS patients. In parallel, median levels of anti-tetanus and anti-HBV IgG were significantly reduced compared to controls [0.05 (0.03-0.16) vs. 0.45 (0.29-3.10) IU/ml and 0.0 (0.0-0.5) vs. 30.3 (5.5-400.8) mIU/ml, respectively; p = 0.02 for both], with serum IgG titers below protective threshold in 7/11 SSNS patients for tetanus and in 9/11 SSNS patients for HBV. In contrast, all SSNS patients had a competent B-cell response, showing an amount of total IgG-secreting B cells >1,000 counts/10⁶ stimulated cells. The amount of anti-tetanus and anti-HBV IgG-secreting B cells was also comparable to that of controls (p = 0.24, p = 0.32, respectively), with a frequency of memory anti-tetanus and anti-HBV IgG secreting B cells >0.1% of total IgG secreting B cells. In conclusion, SSNS children at disease onset pre-immunosuppressive therapy showed a competent immune and vaccine response against tetanus and HBV, which can be correctly evaluated by quantification of antigen-specific memory B cells rather than by measuring serum IgG levels. This approach allows early identification of the impairment of immune and vaccine competence, which may derive from protracted use of different immunosuppressive drugs during disease course.

Keywords: ELISPOT; IgG; immune competence; pediatric nephrology; steroid-sensitive nephrotic syndrome; vaccine competence.

Figure 1

Serum immunoglobulin levels in steroid-sensitive nephrotic syndrome pediatric patients at onset. (A) Levels of total serum IgG, IgA and IgM were measured in steroid-sensitive nephrotic syndrome pediatric patients at disease onset (SSNS, n=10/11) and expressed as mg/dl. In one patient serum immunoglobulin levels were not determined. Each plot represents a different patient. Gray areas represent the age-related normal range as indicated by the diagnostic laboratory of Bambino Gesù Children's Hospital, IRCCS. (B,C) Antigen-specific IgG titers against (B) tetanus and (C) hepatitis B virus (HBV) were measured in SSNS pediatric patients at onset (n=11) and in age-matched controls (CTRL, n=5) and expressed as IU/ml and mIU/ml, respectively. Protective levels identified by dashed gray lines were indicated in the diagnostic laboratory of Bambino Gesù Children's Hospital, IRCCS. Horizontal lines indicate the medians and differences between groups were compared using the Mann–Whitney U test.

Figure 2

Total and antigen-specific IgG-secreting B cells in steroid-sensitive nephrotic syndrome pediatric patients at onset. (A–E) Isolated PBMCs were stimulated for 5 days with CpG plus rhIL-21 and rhIL-4. Following stimulation, (A) total, (B,C) anti-tetanus and (D,E) anti-HBV IgG-secreting B cells were enumerated by ELISPOT in steroid-sensitive nephrotic syndrome pediatric patients at disease onset (SSNS, n=11) and in age-matched controls (CTRL, n=5). Antigen-specific memory B cells were represented as (B,D) absolute count/10⁶ cells and as (C,E) percentage of total IgG-secreting B cells. Each plot represents a different patient. Horizontal lines indicate the means and differences between groups were compared using the unpaired t test.