Human milk antibodies to global pathogens reveal geographic and interindividual variations in IgA and IgG

BACKGROUND. The use of high-throughput technologies has enabled rapid advancement in the knowledge of host immune responses to pathogens. Our objective was to compare the repertoire, protection, and maternal factors associated with human milk antibodies to infectious pathogens in different economic and geographic locations. METHODS. Using multipathogen protein microarrays, 878 milk and 94 paired serum samples collected from 695 women in 5 high and low-to-middle income countries (Bangladesh, Finland, Peru, Pakistan, and the United States) were assessed for specific IgA and IgG antibodies to 1,607 proteins from 30 enteric, respiratory, and bloodborne pathogens. RESULTS. The antibody coverage across enteric and respiratory pathogens was highest in Bangladeshi and Pakistani cohorts and lowest in the U.S. and Finland. While some pathogens induced a dominant IgA response (Campylobacter, Klebsiella, Acinetobacter, Cryptosporidium, and pertussis), others elicited both IgA and IgG antibodies in milk and serum, possibly related to the invasiveness of the infection (Shigella, enteropathogenic E. coli “EPEC”, Streptococcus pneumoniae, Staphylococcus aureus, and Group B Streptococcus). Besides the differences between economic regions and decreases in concentrations over time, human milk IgA and IgG antibody concentrations were lower in mothers with high BMI and higher parity, respectively. In Bangladeshi infants, a higher specific IgA concentration in human milk was associated with delayed time to rotavirus infection, implying protective properties of antirotavirus antibodies, whereas a higher IgA antibody concentration was associated with greater incidence of Campylobacter infection. CONCLUSION. This comprehensive assessment of human milk antibody profiles may be used to guide the development of passive protection strategies against infant morbidity and mortality

Inferring Epitopes of a Polymorphic Antigen Amidst Broadly Cross-Reactive Antibodies Using Protein Microarrays: A Study of OspC Proteins of <i>Borrelia burgdorferi</i>

<div><p>Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming ‘wet bench’ techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and <i>Peromyscus leucopus</i> rodents infected with <i>Borrelia burgdorferi</i> to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed <i>in silico</i> screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants.</p></div

Comparison of conserved and variable residues between OspC pairs A, I3 and F, I3.

<p><b>Panel A</b> Partial amino acid sequence alignment of OspC types F (top row), A (bottom row) and the chimeric OspC I3 (middle). Only helices α2 through α5 are shown. Black bars indicate regions of sequence identity between the chimeric OspC I3 to the parental OspC types. Colored blocks represent individual amino acids. <b>Panel B</b> Top row: frontal view of the OspC dimer structure; bottom row, view from the top of the structure. The cartoon representation of the OspC dimer is colored from N- to C- terminus in blue to red, respectively. The surface representations show the combined amino acid sequences of pairs OspC A and I3 (left) and OspC F and I3 (center), where polymorphic positions identical within each pair are shown in green and mismatches appear in red. Residues strictly conserved in all 23 OspC proteins appear in light and dark gray.</p

Matrices of cross-reactive antibody binding correlations.

<p>Pearson’s correlation (<i>r</i>) was calculated between all OspC pairs using antibody binding reactivity values for all 55 sera from patients with LD to populate the matrices shown as heat maps. The matrices computed for log₁₀-transformed data (<i>M_D-log</i>), rank transform (<i>M_D-rank</i>) and binary transform (<i>M_D-binary</i>) are shown in the left, middle and right panels, respectively.</p

Maximum cross-reactivity regions for individual OspC types.

<p>Solvent-accessible surface area representation of the 3D structure model constructed from the <i>MSA</i> using UCSF Chimera from the structure of OspC-A (pdb 1GGQ). The chains of OspC dimer are colored light and dark grey. The location of residues of highest correlation (<i>r</i> value) with antibody cross-reactivity (<i>M_D-avg3</i>) are colored as follows: highest <i>r</i> value in one OspC protein (green); in two OspC proteins (yellow); in five OspC proteins (red).</p

Heat maps of correlation between local sequence identity using subsets of sequentially consecutive polymorphic positions and antibody cross-reactivity.

<p>Local sequence regions were systematically defined and correlations were calculated as described in Methods for Sequence Scanning. Each panel shows results from the calculation performed using the 3 transforms: log-transformed, rank and binary, on the left, middle and right panels, respectively. Each row in a heat map corresponds to the center of a sequence window and the columns encompass the results calculated using window sizes varying from 3 to 116 residues (only polymorphic positions are considered in the size). The individual alpha helical structures are indicated α1 through α5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067445#pone.0067445-Kumaran1" target="_blank">[50]</a>. The green to red gradient bar indicates the range of <i>r</i> values observed in the results (min: −0.33; max: 0.39).</p

Twenty most cross-reactive OspC types for sera from patients with Lyme disease.

<p>Twenty most cross-reactive OspC types for sera from patients with Lyme disease.</p

Distribution of Pearson’s <i>r values</i> from OspC cross-reactivity matrices.

<p>The frequency histogram shows the distribution of <i>r</i> values obtained from the OspC cross-reactivity matrices calculated using either the observed (blue bars) or randomized (orange bars) antibody binding profiles and their 3 transform metrics, log₁₀, rank and binary.</p

Multivariate analysis of antibody response to OspC types A, F and the chimera I3.

<p>Log10-transformed intensity of antibody binding to OspC A, F and I3 by sera from 55 human patients with LD (blue dots) and 23 experimental infections of <i>P. leucopus</i> rodents (red dots). Correlation coefficient <i>R²</i> is shown for each comparison.</p