Serum IgG Profiling of Toddlers Reveals a Subgroup with Elevated Seropositive Antibodies to Viruses Correlating with Increased Vaccine and Autoantigen Responses

Abstract

Purpose: The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses.

Methods: An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses.

Results: IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (17%) had elevated IgG responses to multiple RNA and DNA viruses (e.g., respiratory syncytial virus, Epstein-Barr virus, adenovirus, Coxsackievirus) and this correlated with increased responses to live attenuated viral vaccines and certain autoantigens. This high group was more likely to be associated with gestational diabetes and an older age. Genetic analyses identified polymorphisms in the IL2RB, TNFSF4, and INS genes in two high responder individuals that were associated with their elevated cytokine levels and clinical history of eczema and asthma.

Conclusion: Serum IgG profiling of toddlers reveals correlations between the magnitude of the antibody responses towards viruses, live attenuated vaccines, and certain autoantigens. A low responder group had much weaker responses overall, including against vaccines. The serum antibody screen also identifies individuals with IgG responses to less common infections (West Nile virus, parvovirus, tuberculosis). The characterization of the antibody responses in combination with the identification of genetic risk alleles provides an opportunity to identify children with increased risk of clinical disease.

Keywords: IgGs; antigen arrays; serum antibody responses; toddler immune responses; vaccines.

Fig. 1

The serum IgG specificities of toddlers/children reveal distinct subgroup responses to pathogens, autoantigens, vaccines, and allergens. a Schematic of the immune response profiling of 1- and 2-year-old toddlers. Blood samples are processed for serum and cellular DNA, used for antigen arrays, ELISA assays, and DNA sequencing. Medical records are reviewed along with family history. b The antigen array was used to assess the antibody responses in a cohort of 1- and 2-year toddlers/children. One hundred ninety-seven individual serum samples were tested against an antigen array comprising 110 antigens (detailed in the Methods section). The heat maps, with IgM on the left and IgG on the right, correspond to 197 samples; each column is 1 sample/toddler. The various categories of antigens are indicated. The heat map indicates positive antibody reactivity (red color) or minimal to no reactivity (green color). c The sum of the IgG nfi responses for every antigen was calculated, revealing a segregation of the cohort into low, moderate (Mod), and high reactivity groups. d The relative serum IgG level for each individual, stratified into the low, Mod, and high response group, was determined. e The nfi levels for hen egg lysozyme (HEL), used as a control antigen, were determined for each individual in the low, Mod, and high response groups. Statistical analyses were done with one-way ANOVA using Tukey’s correlation, and p values shown. ns, not significant

Fig. 2

Serum IgG nfi seropositive responses to particular RNA and DNA viruses cluster among the high responder group. a The relative IgG responses, measured using the nfi values from the microarray analysis, are shown for two distinct RSV antigens, shown as black (RSV) and red lines (RSV glycoprotein, RSV-Gp). Each line corresponds to an individual sample, ranged from the lowest to highest overall nfi value. The separation among the low, Mod, and high groups is shown below the y-axis. Serum IgG responses, determined using an independent ELISA assay for an RSV-Gp, are shown for a subset of toddlers. The adjusted OD₄₅₀ values are shown as open circles. b The relative IgG nfi responses are shown for two distinct CMV antigens, shown with black (CMV) and red lines (CMV-M). An independent ELISA assay for the CMV antigen was performed using samples from a subset of toddlers. The open circles reveal the adjusted OD₄₅₀. c–h Serum IgG nfi values specific for c adenovirus, d coronavirus, e Coxsackievirus, f echovirus type 9, and f enterovirus 71 are shown, plotted against the increasing overall nfis calculated for each individual. Values above the dotted lines in the graphs suggest infection-based seropositivity

Fig. 3

Serum IgG nfi values are elevated towards RNA and DNA viruses and live attenuated viral vaccines in a subset of toddlers. a The relative IgG responses (nfis) are shown for various RNA viruses (red), DNA viruses (blue), bacterial antigens (black), and one parasite, as indicated. These values are shown based on the stratification of the toddler groups into low, Mod, and high. The high responder group exhibited a statistically significant increase in the IgG nfi values towards selected viral pathogen antigens, as shown. This included responses to RSV, Coxsackievirus, echovirus type 9, CMV type 3 antigen, and adenovirus type 5. b The relative IgG responses to live attenuated viruses, inactivated viruses, or subunit vaccines were determined. As in a, these values are shown relative to the stratified groups defined as low, Mod, and high. Statistical analyses were done with one-way ANOVA using Tukey’s correlation, with the calculated p values shown. ns, not significant

Fig. 4

Responses to selected autoantigens were greater in the high reactivity group a. The relative IgG responses (nfi values) are shown for diverse autoantigens among the low, Mod, and high groups. The high responder group exhibited a statistically significant increase in the IgG nfi values towards nucleosome components, arrestin b1, a histone mix, endothelial cell lysates, and the Smith antigens (Sm/Smd). b Pearson’s correlation analysis was performed for the sum of the total nfis for each of the 197 samples in relation to the summed nfis for 7 viruses (parvovirus, enterovirus 71, adenovirus type 5, RSV, Coxsackievirus, echovirus type 9, CMV), 5 live attenuated vaccines (mumps, rotavirus, rubella, rubeola, varicella-zoster), 7 subunit vaccines (hepatitis B, hepatitis A, Haemophilus influenzae, inactivated influenza types A and B, tetanus toxoid, Streptococcus pneumoniae), and 8 autoantigens (arrestin beta 1, histones, myelin basic protein, myosin, endothelial lysates, Smith antigens, nucleosomes, alpha fodrin). Statistical analyses were done with Pearson correlation calculations using Graph Prism 8. The r value and the R² values are indicated within the graphs. c The serum samples from a subset of toddlers (114/197) were used in ELISA assays to screen for the presence of anti-nuclear antibodies (ANA). The cutoff for positivity was defined by a previous analysis of over 700 samples from SLE patients, with positive controls provided within the ANA kit (ANA kit, QUANTA Lite ANA ELISA by Inova Diagnostics). Thirty (26%) and eight (7%) of these samples had moderate and high ANA titers. d The low, Mod, and high reactivity groups were compared for their ANA levels. Eight with high ANAs were part of the high reactivity group. e Odds ratio comparing the high responder cohort and diverse clinical indicators. The clinical information from 110 low and 33 high responder cohorts was compared to determine the relative odds ratio and statistical significance with particular clinical conditions. The strongest relationships exist with gestational diabetes followed by age. Statistical analyses were done with one-way ANOVA using Tukey’s correlation, with the calculated p values shown. ns, not significant. For odds ratio calculations, 2-way calculations were performed with the 95% confidence intervals shown

Fig. 5

Longitudinal profiling of serum IgG nfi responses in toddlers/children from year 1 to year 2 reveals both fixed and fluctuating patterns dependent on the antigen. a The relative IgG responses (nfi values) are shown against various RNA viruses (red), DNA viruses (blue), and a bacterial antigen (black) as indicated. These values are shown for 41 repeat samples, using a serum sample from Yr.1 followed by a second sample at Yr.2. The high responder group exhibited a statistically significant increase in the IgG nfi values towards selected viral pathogen antigens, as shown. This included responses to RSV, Coxsackievirus, echovirus type 9, CMV, and adenovirus type 5. b The relative IgG responses to either live attenuated, inactivated, or subunit vaccines were determined with the 41 repeats. Statistical analyses were done with one-way ANOVA using Tukey’s correlation, with the calculated p values shown. ns, not significant

Fig. 6

Correlation between genetic risk alleles and high responder cohort. a Schematic approach for identifying SNPs in immune response genes in the toddlers/children that have been linked to various clinical conditions including asthma, celiac, Crohn’s disease, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), lupus (SLE), sclerosis, type I diabetes, and ulcerative colitis. b The sum total of each of the 47 genetic risk alleles (94 total when considering both alleles) was determined for 114 individual toddlers. These values were plotted against the low (n = 110), Mod (n = 54), and high (n = 33) responder cohorts. c Pearson’s correlation analysis was performed for the sum of the total genetic risk alleles for 114 samples in relation to the summed IgG nfis towards the 110 distinct antigens. Statistical analysis was done by Pearson’s correlation. The r value and the R² values are indicated within the graph. d The low and high genetic risk scores were compared within the 1st year age group and the 2nd year age group

Fig. 7

DNA sequence polymorphisms in immune responses genes linked to elevated serum cytokine levels in toddlers. a–c The total sum of the IgG nfi responses for every antigen was calculated. These values were plotted against the allelic polymorphisms in the cohort for the genes: a IL2 receptor beta gene (rs2284033), b TNFSF4 (rs7514229), and c IGF2 (rs689). Two of the toddlers in the high response group (72.1 and 147.1) are indicated with the red colored circles or boxes. The two from the low response group (131.1, 141.1) are in blue. d Four serum samples, two from the IL2rb (rs2284033) A/G haplotype (132.1, 141.1) and two from G/G genotype (147.1, 72.1), were analyzed with the Human 48 Plex BioRad cytokine/chemokine assay. A heat map was used to illustrate the levels of inflammatory cytokines and chemokines present in each sample. Two individuals, who are in the high responder categories, had markedly elevated levels of the indicated cytokines (147.1, 72.1)